Dermal micro-organs, methods and apparatuses for producing and using the same

ABSTRACT

Embodiments of the present invention provide Dermal Micro-organs (DMOs), methods and apparatuses for producing the same. Some embodiments of the invention provide a DMO including a plurality of dermal components, which substantially retain the micro-architecture and three dimensional structure of the dermal tissue from which they are derived, having dimensions selected so as to allow passive diffusion of adequate nutrients and gases to cells of the DMO and diffusion of cellular waste out of the cells so as to minimize cellular toxicity and concomitant death due to insufficient nutrition and accumulation of waste in the DMO. Some embodiments of the invention provide methods and apparatuses for harvesting the DMO. An apparatus for harvesting the DMO to may include, according to some exemplary embodiments, a support configuration to support a skin-related tissue structure from which the DMO is to be harvested, and a cutting tool able to separate the DMO from the skin-related tissue structure. Other embodiments are described and claimed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.12/572,013, filed on Oct. 1, 2009, which is a Continuation in Part ofU.S. application Ser. No. 12/216,321, filed on Jul. 2, 2008, which is aContinuation of U.S. application Ser. No. 10/834,345, filed Apr. 29,2004, now U.S. Pat. No. 7,468,242, which claims priority from U.S.Provisional Application No. 60/466,793, filed May 1, 2003, and U.S.Provisional Application No. 60/492,754, filed Aug. 6, 2003 and is a toContinuation in Part of PCT International Application NumbersPCT/IL02/00877, PCT/IL02/00878, PCT/IL02/00879 and PCT/IL02/00880, allfiled Nov. 5, 2002, which claim priority from U.S. ProvisionalApplication No. 60/330,959, filed Nov. 5, 2001, U.S. ProvisionalApplication No. 60/393,745, filed Jul. 8, 2002 and U.S. ProvisionalApplication No. 60/393,746, filed Jul. 8, 2002, the disclosures of allof which are incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to the field of tissue based micro-organs,therapeutic tissue based micro-organs and methods and apparatuses forharvesting, processing, implanting and manipulating dermal tissue.

BACKGROUND OF THE INVENTION

Various methods for delivering therapeutic agents are known. Forexample, therapeutic agents can be delivered orally, transdermally, byinhalation, by injection and by depot with slow release. In each ofthese cases the method of delivery is limited by the body processes thatthe agent is subjected to, by the requirement for frequentadministration, and limitations on the size of molecules that can beutilized. For some of the methods, the amount of therapeutic agentvaries between administrations.

A dermal micro-organ (DMO), which can be sustained outside the body (“exvivo” or “in vitro”) in an autonomously functional state for an extendedperiod of time, and to which various manipulations can be applied, maythen be implanted subcutaneously or within the body for the purpose oftreating diseases, or disorders, or for plastic surgical purposes. TheDMO can be modified to express a gene product of interest. Thesemodified dermal micro-organs are generally referred to as DermalTherapeutic Micro-Organs (DTMOs).

Skin micro-organs, including layers of epidermal and dermal tissues, forexample; as outlined in PCT/IL02/0880, have been observed to beassociated with a number of clinical challenges. Harvesting of a skinsample leaves a superficial wound on the patient that may last severalweeks and may leave scars. The harvested skin sample requiressignificant processing to generate micro-organs from this sample. Also,implantation of skin micro-organs subcutaneously or to deeper in thebody have been found to result in the development of keratin cysts orkeratin micro-cysts. Additionally, implantation of skin micro-organs asa graft onto the skin surface in “slits” requires significant technicalexpertise in order to handle the MO while maintaining its properorientation.

Harvesting of dermis, e.g., to be used as a “filler material” in aplastic surgical or cosmetic procedure, is known in the art.Conventional harvesting techniques include using a dermatome or scalpelto peel away a layer of epidermis in order to expose a section ofdermis. The dermatome or scalpel may then be used again to manuallyharvest the exposed section of dermis.

Another conventional apparatus for harvesting dermis, albeit notcommonly used, is the Martin Dermal Harvester marketed by Padgett (PartNo. P-225) for the indication of harvesting dermal cores from the backfor subsequent implantation into the lips during cosmetic lipaugmentation procedures. To operate this device, which is not commonlyused, a sharpened cutting tube, which includes a reusable thick walledtube with an inner diameter of approximately 4.5 mm, is manually rotatedat a very slow speed. Using this type of device generally requiresapplying pressure to the skin surface directly above the harvest siteand installing sutures with active tugging as the cutting tube is pushedforward. Furthermore, the resulting harvested dermis is generally notuniform in dimensions and includes “plugs” of epidermis at either end ofthe dermal core.

SUMMARY OF THE INVENTION

Embodiments of some aspects of the present invention provide a DMO/DTMOwith the ability to be maintained ex-vivo in a generally viable state,which may allow various manipulations to be performed on the DMO, whilekeeping a high production and secretion level of the desired therapeuticagent. In addition, embodiments of some aspects of the present inventionprovide a method of harvesting a DMO and subsequently implanting a DTMOwithout forming keratin cysts or keratin microcysts, e.g., uponimplantation of the DTMO subcutaneously or deeper in the body.Furthermore, it will be appreciated by persons skilled in the art thatthe methods and devices according to some embodiments of the presentinvention may be relatively uncomplicated and, therefore, the level ofskill required from a professional to carry out the methods and/or touse the devices of the present invention may not be as demanding asthose required in conventional procedures.

Some exemplary embodiments of the invention provide a dermal micro-organ(DMO) to having a plurality of dermal components, which may includecells of the dermal tissue and a surrounding matrix. The DMO accordingto embodiments of the invention may generally retain amicro-architecture and three dimensional structure of the dermal organfrom which it is obtained and the dimensions of the DMO may allowpassive diffusion of adequate nutrients and gases to the cells anddiffusion of cellular waste out of the cells so as to minimize cellulartoxicity and concomitant death due to insufficient nutrition andaccumulation of waste.

In some exemplary embodiments of the invention, the dermal micro-organof the invention does not produce keratin or produces negligible amountsof keratin.

In some embodiments of the invention, the dermal micro-organ does notproduce keratin and/or keratin cysts following subcutaneous or deeperimplantation in a body.

In another embodiment of the invention, the dermal micro-organ of theinvention produces micro keratin cysts following that will atrophywithin a relatively short period of time, e.g., days or weeks aftersubcutaneous implantation.

In another embodiment of the invention, the dermal micro-organ of theinvention contains hair follicles and sebaceous glands, which willatrophy after a short period of time, e.g., days or weeks.

In another embodiment of the invention, the dermal micro-organ of theinvention contains glands that will connect to the skin surface after ashort period of time, e.g., days or weeks.

Further exemplary embodiments of the invention provide a method andapparatus of harvesting a dermal micro-organ. The method may includestabilizing and/or supporting a skin-related tissue structure from whicha dermal micro-organ is to be harvested, e.g., such that theskin-related tissue structure is maintained at a desired shape and/orposition, separating at least a portion of the dermal micro-organ fromthe skin-related tissue structure, and isolating the separated dermalmicro-organ from the body. According to some of these exemplaryembodiments, the support configuration may include a first tubularelement, and the cutting tool may include a second tubular elementadapted to be inserted along and substantially coaxially with the firstelement. According to other exemplary embodiments, the supportconfiguration may include a vacuum chamber having an inner supportsurface able to maintain the skin-related tissue structure at a desiredshape and/or position to enable the cutting tool to separate the DMOfrom the skin-related tissue structure.

Further exemplary embodiments of the invention provide a geneticallymodified dermal micro-organ expressing at least one recombinant geneproduct the dermal micro-organ having a plurality of dermal components,including cells and matrix of the dermal tissue, which retain themicro-architecture and three dimensional structure of the dermal tissuefrom which they are obtained, and having dimensions selected so as toallow passive diffusion of adequate nutrients and gases to the cells anddiffusion of cellular waste out of the cells so as to minimize cellulartoxicity and concomitant death due to insufficient nutrition andaccumulation of waste, wherein at least some of the cells of the dermalmicro-organ express at least one recombinant gene product or at least aportion of said at least one recombinant gene product.

In some embodiments of the invention, the recombinant gene product is ablood clotting factor.

In some embodiments of the invention, the recombinant gene product isFactor VIII.

In some embodiments of the invention, the recombinant gene product isFactor IX.

Yet further exemplary embodiments the invention provide a geneticallymodified dermal micro-organ expressing at least one recombinant protein,the dermal micro-organ having a plurality of dermal components,including cells and matrix of the dermal tissue, which retain themicro-architecture and three dimensional structure of the dermal tissuefrom which they are obtained, and having dimensions selected so as toallow passive diffusion of adequate nutrients and gases to the cells anddiffusion of cellular waste out of then cells so as to minimize cellulartoxicity and concomitant death due to insufficient nutrition andaccumulation of waste, wherein at least some of the cells of the dermalmicro-organ express at least a portion of at least one recombinantprotein.

In some embodiments of the invention, the recombinant protein is a bloodclotting factor.

In some embodiments of the invention, the recombinant protein is FactorVIII.

In some embodiments of the invention, the recombinant protein is FactorIX.

In some embodiments of the invention, the genetically modified dermalmicro-organ of the invention produces substantially no keratin.

In some embodiments, the invention provides a method of delivering to arecipient a recombinant gene product produced by the dermal micro-organ.

In some embodiments, the invention provides a method of inducing a localor systemic physiological effect by implanting a dermal micro-organ in arecipient.

In another embodiment the invention provides a method of delivering aprotein of interest to a subject. The method includes implanting thegenetically modified dermal micro-organ into the skin, under the skin orat other locations in the body.

In another embodiment, the invention provides a method of implanting adermal micro-organ so as to avoid or to reduce keratin cyst formation.

BRIEF DESCRIPTION OF DRAWINGS

Non-limiting embodiments of the invention are described in the followingdescription, to be read with reference to the figures attached hereto.In the figures, identical and similar structures, elements or partsthereof that appear in more than one figure are generally labeled withthe same or similar references in the figures in which they appear.Dimensions of components and features shown in the figures are chosenprimarily for convenience and clarity of presentation and are notnecessarily to scale.

FIG. 1 is a schematic block diagram of an exemplary method of producingand utilizing dermal therapeutic micro-organs (DTMOs), in accordancewith an exemplary embodiment of the invention;

FIGS. 2A and 2B show, respectively, a correlation analysis betweenin-vitro secretion of pre-implanted mIFN alpha.-TMOs and hEPO-TMOs andthe serum in-vivo levels following their implantation, in accordancewith an embodiment of the invention;

FIGS. 3A and 3B show, respectively, elevated serum hEPO levelsdetermined by an ELISA assay and reticulocyte count elevation afterautologous TMO implantation in a miniature swine, in accordance with anembodiment of the invention;

FIG. 4 is a schematic illustration of a graph showing secretion levelsof human erythropoietin (hEPO) by DTMO-hEPO prepared from six differenthuman skins;

FIG. 5 shows histology of DTMO and split thickness skin TMO;

FIG. 6 shows Immunohistochemistry (IHC) and Hematoxylin & Eosin (H&E)staining of DTMO;

FIG. 7 demonstrates in vivo hEPO serum levels and physiological effecton hematocrit levels following subcutaneous implantation of DTMO-hEPOand split thickness skin TMO-hEPO in SCID mice;

FIG. 8 demonstrates clinical and histological analysis of DTMO-hEPO andsplit thickness skin TMO-hEPO implanted subcutaneously in SCID mice;

FIG. 9 shows Histological analysis of skin MOs grafted in skin slits(split thickness skin MO, right) or implanted S.C. (DMO, Left) 17 dayspost implantation in healthy volunteers;

FIG. 10 is a schematic flowchart illustrating a method of harvesting aDMO according to some exemplary embodiments of the invention;

FIGS. 11 a-11 c are schematic illustrations of exemplary stages ofharvesting a DMO in accordance with the method of FIG. 10;

FIG. 12 is a schematic illustration of a clamping tool that may be usedby a dermal harvesting apparatus in accordance with some exemplaryembodiments of the invention;

FIG. 13 is a schematic illustration of a dermal harvesting apparatusincluding a coring tube inserted into source tissue for a DMO, andharvesting coaxially with an inner guide needle in accordance with someexemplary embodiments of the invention;

FIGS. 14 a-14 c are schematic illustrations of a front view, a sideview, and top view, respectively, of a dermal vacuum harvestingapparatus according to an exemplary embodiment of the invention;

FIG. 15 is a schematic illustration of a cross-sectional side view ofthe apparatus of FIGS. 14 a-14 c supporting a dermal micro-organ at adesired position according to one exemplary embodiment of the invention;

FIG. 16 is a schematic illustration of a cross-sectional view of theapparatus of FIG. 15 externally supporting a dermal micro-organ to beharvested at a desired position;

FIG. 17 is a schematic illustration of a dermal harvesting apparatusaccording to another exemplary embodiment of the invention;

FIG. 18 is a schematic illustration of a harvesting apparatus accordingto yet another exemplary embodiment of the invention;

FIG. 19 is a schematic illustration of implementing the harvestingapparatus of FIG. 18 for harvesting a DMO;

FIG. 20 is a flow chart illustrating a DTMO implanting method, accordingto some embodiments of the present invention;

FIG. 21 is a flow chart illustrating a DTMO ablating method, accordingto some embodiments of the present invention; and

FIG. 22 is a schematic illustration of a system for processing aharvested DMO according to exemplary embodiments of the invention.

FIG. 23 is a schematic illustration of a graph showing secretion levelsof human erythropoietin expressed from either a wild-type gene sequenceor optimized gene sequence.

FIG. 24 is a schematic illustration of a graph showing secretion ofalpha-interferon over an extended time period.

FIG. 25 is a schematic illustration of a graph showing high levels ofsecretion of alpha-1-antitrypsin.

FIG. 26 is a schematic illustration of a graph showing increased levelsof erythropoietin secretion in the presence of cis-acting S/MARelements.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The following description is presented to enable one of ordinary skillin the art to make and use the invention as provided in the context of aparticular application and its requirements. Various modifications tothe described embodiments will be apparent to those with skill in theart, and the general principles defined herein may be applied to otherembodiments. Therefore, the present invention is not intended to belimited to the particular embodiments shown and described, but is to beaccorded the widest scope consistent with the principles and novelfeatures herein disclosed. In other instances, well-known methods,procedures, and components have not been described in detail so as notto obscure the present invention.

In the following detailed description, numerous specific details are setforth in order to provide a thorough understanding of the presentinvention. However, it will be understood by those skilled in the artthat the present invention may be practiced without these specificdetails.

Exemplary Definitions of Terms Used Herein

The term “explant” as used herein, refers in some embodiments of theinvention, to a removed section of living tissue or organ from one ormore tissues or organs of a subject.

The term “dermal micro-organ” or “DMO” as used herein, refers in someembodiments of the invention, to an isolated tissue or organ structurederived from or identical to an explant that has been prepared in amanner conducive to cell viability and function, while maintaining atleast some in vivo interactions similar to the tissues or organ fromwhich it is obtained. Dermal micro-organs may include plurality ofdermal components that retain the micro-architecture of the tissue ororgan from which they were derived, and three dimensional structure ofthe dermal tissue from which they are derived, having dimensionsselected so as to allow passive diffusion of adequate nutrients andgases to cells within the MO and diffusion of cellular waste out of thecells of the MO so as to minimize cellular toxicity and concomitantdeath due to insufficient nutrition and accumulation of waste. Dermalmicro-organs may consist essentially of a plurality of dermis components(tissue components of the skin located below the epidermis). Thesecomponents may contain skin fibroblast, epithelial cells, other celltypes, bases of hair follicles, nerve endings, sweat and sebaceousglands, and blood and lymph vessels.

Wherever used herein below, the description of the embodiments relatedto MO relates also to dermal MO whenever the term “dermal tissue” isused, it also relates to “dermal organ”.

As used herein, the term “microarchitecture” refers, in some embodimentsof the invention, to the characteristic of the explant in which, in oneembodiment at least about 50%, in another to embodiment, at least about60%, in another embodiment at least about 70%, in another embodiment, atleast about 80%, and in another embodiment, at least about 90% or moreof the cells of the population, maintain, in vitro, their physicaland/or functional contact with at least one cell or non-cellularsubstance with which they were in physical and/or functional contact invivo. Preferably, the cells of the explant maintain at least onebiological activity of the organ or tissue from which they are isolated.

The term “donor” as used herein, refers in some embodiments of theinvention to a subject, from which the explant is removed and used toform, or which is already in the form of, one or more micro-organs.

The term “therapeutic micro-organ (TMO)” as used herein, refers in someembodiments of the invention to a micro-organ (MO) that can be used tofacilitate a therapeutic objective, such as, for example, an MO that hasbeen genetically altered or modified to produce a therapeutic agent,such as a protein or and RNA molecule. The therapeutic agent may or maynot be a naturally occurring body substance. Wherever used hereinbelow,the description of the embodiments related to TMO relates also to DTMOwhich is a therapeutic Dermal MO which may be in some embodiments of theinvention genetically modified.

The term “implantation” as used herein, refers in some embodiments ofthe invention, to introduction of one or more TMOs or DTMOs into arecipient, wherein said TMOs or DTMOs may be derived from tissues of therecipient or from tissues of another individual or animal. The TMOs orDTMOs can be implanted in a slit within the skin, by subcutaneousimplantation, or by placement at other desired sites within therecipient body.

The term “recipient” as used herein refers, in some embodiments of theinvention, to a subject, into which one or more TMOs or DTMOs areimplanted.

The term “clamping” (e.g., the skin) as used herein may refer to anysimilar action or any action with a similar purpose, for example,“pinching” (e.g., the skin).

The term “in vitro” as used herein should be understood to include“ex-vivo”.

The term “coring tube” as used herein may relate, individually orcollectively, to the terms “cutting tool”, “cutting tube” and “coringneedle”, as well as to any other elements with similar functionalities.

While, for clarity and completeness of presentation, all aspects of theproduction and utilization of DTMOs are described in this document, andembodiments of the invention are described from the start of theprocesses to their ends, it should be understood that each of theaspects described herein can be used with other methodologies and/orequipment for the carrying out of other aspects and can be used forother purposes, some of which are described herein. The presentinvention includes portions devoted to the preparation and maintenanceof dermal micro-organs for transformation into DTMOs. It should beunderstood that the dermal micro-organs produced according to theseaspects of the invention can be used for purposes other than fortransformation into DTMOs

In some embodiments of the invention, the micro-organ is a dermalmicro-organ including a plurality of dermis components, for example,fibroblasts and/or epithelial components containing nerve endings and/orsweat glands and/or sebaceous glands and/or blood and lymph vesselsand/or elastin fibers and/or collagen fibers and/or endothelialcomponents and/or immune system derived cells and/or extra-cellularmatrix. As shown by the test results summarized in the Examples sectionbelow (Example 5, FIG. 8), conventional subcutaneous implantation of amicro-organ including epidermal layers (“split thickness skin MO”) inmice and pigs (data in pigs is not shown), may result in formation ofkeratin cysts or macro-keratin cysts. In contrast, when skin tissue issampled to obtain a DMO according to exemplary embodiments of theinvention, no cysts or macro cysts are observed in mice, pigs or inhumans. It should be noted that the biological activity (for example,secretion of a therapeutic protein, e.g., erythropoietin and elevationof hematocrit as a result) of a DTMO according to embodiments of theinvention may be comparable to or even higher than split thickness skinderived TMO (see Example 4). Namely, both types of preparation mayrelease the same amount of erythropoietin; however, the DTMO may produceand secrete higher protein levels per unit than those of split thicknessderived TMO.

In general, production of DTMOs may include DMO harvesting, maintainingthe DMO and/or modifying the DMO and/or genetically altering them and,in some embodiments, verifying the production of a desired agent (forexample proteins) by the DMO. Utilization of the DTMO may includeproduction, within a patient's or animal's own body, of therapeuticsubstance, such as proteins, for treatment of a subject. For example,the DTMO can be implanted into or under the skin or within the body ofthe subject to produce the agent/protein in vivo. In the case of tissuefrom another subject, the implant is optionally protected from reactionby the recipient's immune system, for example, by housing the DTMO in animmunoprotective capsule or sheath. For example, a membrane can bepositioned to surround the DTMO, either by placing the DTMO in a capsuleprior to implantation or otherwise. The membrane should have a pore sizethat is sufficiently large to allow for the passage of nutrients, wasteand the therapeutic agent yet sufficiently small to prevent passage ofcells of the immune system.

In some embodiments of the invention, the dermal micro-organ may containtissue of a basal epidermal layer and, optionally, other epidermallayers of the skin. In other embodiments, the dermal micro-organ doesnot include basal layer tissue.

In some embodiments of the invention, the DMO does not include epidermallayers. In other embodiments, the DMO may contain a few layers ofepidermal tissue.

In one embodiment of the invention, the DMO includes the entirecross-section of the dermis.

In another embodiment of the invention, the dermal micro-organ includespart of the cross-section of the dermis. In a further embodiment, theDMO includes most of the cross section of the dermis, namely, most ofthe layers and components of the dermis including the papillary andreticular dermis. In a further embodiment, the DMO includes primarilydermal tissue, but may also include fat tissue. In some embodiments ofthe invention, the DMO does not produce keratin or produces a negligibleamount of keratin, thereby preventing the formation of keratin cystsfollowing subcutaneous implantation in a recipient.

The DMO to be harvested can be removed from the body by any means ofremoving tissue known in the art, such as biopsy procedures. Theharvesting procedure keeps intact the micro-architecture of the tissuefrom which it is removed. In one embodiment the DMO may be obtained bydirect biopsy and be then cut to the required size or have non-desiredtissue cut from it. In another embodiment, a tissue sample may beobtained by direct biopsy, in which the desired size of the dermalmicro-organ is obtained and no further processing is required.

In some embodiments of the invention, the dermal micro-organ is directlyharvested from the body, and the dimensions of a cutting tool used toharvest the dermal micro-organ may be, for example, about 1-4 mm indiameter. In another embodiment, the dimension may be, for example, 1.71mm in diameter. In another embodiment the dimension may be, for example,1-3 mm in diameter. In another embodiment, the dimension may be, forexample, 2-4 mm in diameter. In another embodiment the dimension may be,for example, 1-2 mm in diameter. In another embodiment the dimension mayto be, for example, about 1.5 mm in diameter. In another embodiment, thedimension may be, for example, about 2 mm in diameter. In someembodiments, the harvested dermal micro-organ may not retain itscylindrical shape after harvesting, i.e., at least one dimension of itscross section may expand while at least another dimension of its crosssection may contract. In one embodiment, for example, at least onedimension may be 0.5-3.5 mm and at least one dimension may be 1.5-10 mm.

In another embodiment, the dimensions of the tissue being harvested maybe, for example, about 5-100 mm in length. In another embodiment, thedimensions of the tissue being harvested may be, for example, about10-60 mm in length. In another embodiment, the dimensions of the tissuebeing harvested may be, for example, about 20-60 mm in length. Inanother embodiment, the dimensions of the tissue being harvested may be,for example, about 20-50 mm in length. In another embodiment, thedimensions of the tissue being harvested may be, for example, about20-40 mm in length. In another embodiment, the dimensions of the tissuebeing harvested may be, for example, about 20-100 mm in length. Inanother embodiment, the dimensions of the tissue being harvested may be,for example, about 30-100 mm in length. In another embodiment, thedimensions of the tissue being harvested may be, for example, about40-100 mm in length. In another embodiment, the dimensions of the tissuebeing harvested may be, for example, about 50-100 mm in length. Inanother embodiment, the dimensions of the tissue being harvested may be,for example, about 60-100 mm in length. In another embodiment, thedimensions of the tissue being harvested may be, for example, about70-100 mm in length. In another embodiment, the dimensions of the tissuebeing harvested may be, for example, about 80-100 mm in length. Inanother embodiment, the dimensions of the tissue being harvested may be,for example, about 90-100 mm with an aspect of some embodiments of theinvention, a closed, sterile, bioreactor apparatus may be used to carry,support and/or alter the DMO or DTMO throughout a harvesting, mm inlength. In another embodiment the length may be around 20 mm. In anotherembodiment, the length may be about 30 mm. In another embodiment, thelength may be about 40 mm.

When a dermal MO has the above listed dimensions, it maybe maintained invitro, e.g., in a growth medium under proper tissue culture conditionsfor extended periods of time, for example, several days, several weeksor several months. The DMO may be maintained, for example, in-vitro indefined growth media. In one exemplary embodiment the growth media mayinclude growth factors, fetal calf serum (FCS), or human serum, e.g.,Synthetic Serum Substitute (SSS). In another exemplary embodiment thegrowth media may include serum either from the donor or the recipientsubject. In yet another embodiment the growth media may includeautologous serum.

In accordance with an aspect of some embodiments of the invention, aclosed, sterile, bioreactor apparatus may be used to carry, supportand/or alter the DMO or DTMO throughout a harvesting, alteration andimplantation process, e.g., from harvesting to implantation, asdescribed in detail below, e.g., with reference to FIG. 22. According tosome exemplary embodiments, at least part of the bioreactor apparatusmay be formed of disposable material.

In accordance with an aspect of some embodiments of the invention, thebioreactor apparatus may be loaded into a docking station, which may beused to carry out various processes and/or to maintain the DMO/DTMOunder desired conditions. The apparatus may be optionally computercontrolled according to a protocol.

In accordance with an aspect of some embodiments of the invention, onlya portion of the DTMO generated may be used in a given treatmentsession. The remaining DTMO tissue may be returned for maintenanceand/or may be stored (e.g., cryogenically or otherwise) for later use.

It is a feature of some embodiments of the invention that a large numberof dermal micro-organs may be processed together in a batch process intoDTMOs, as described below. This may allow for more convenientprocessing, but will not allow for determination of the secretion levelof each DTMO separately.

In some exemplary embodiments of the invention a potency assay may beperformed for the therapeutic agent, which may be produced and/orsecreted by either a single DTMO or a batch of DTMOs. The potency assaymay include, for example, a cell proliferation assay in which theproliferation response of the cells is mainly dependent on the presenceof the therapeutic agent in the growth media of the cells.

The term “skin-related tissue structure”, as used herein, refers to astructure of tissue components that may be stabilized and/or supportedby apparatuses defined herein to enable the harvesting of a dermalmicro-organ therefrom. A skin-related tissue structure may includecomponents of the epidermal tissue, and components of the dermal tissue.Optionally, the skin-related tissue structure may include fat tissueand/or muscle tissue in the vicinity of the dermal tissue.

According to some embodiments of the invention, a method of harvestingthe dermal micro-organ may include stabilizing and supporting askin-related tissue structure from which a dermal micro-organ is to beharvested, e.g., such that at least the dermal micro-organ and/or one ormore other tissue segments in its vicinity are maintained at a desiredshape and/or position, separating at least a portion of the dermalmicro-organ from surrounding tissue, and extracting the separated dermalmicro-organ, as described in detail below.

FIG. 1 shows an overview of a methodology 200 for producing andutilizing DMOs and DTMOs, in block diagram form, in accordance with anexemplary embodiment of the invention. At block 202 a DMO is harvestedfrom a subject. In some embodiments of the invention, the DMO isharvested from the same subject to which therapy will later be applied.In an embodiment of the invention, the DMO is from dermal tissue.Optionally, other tissues are harvested and used in a manner similar tothat described below with reference to dermal tissue. While the methoddescribed below is exemplary, other methods of harvesting tissue samplescan be used in some embodiments of the invention. If desired, the DMOcan be cryogenically stored for later use (i.e., introduction at thesame stage of the process). Alternatively, for certain embodiments, theDMO can be implanted directly back into the patient from which it washarvested to produce a therapeutic, cosmetic, or other physiologicalaffect.

In order for a DMO to be a viable micro-organ, it must have at least onedimension that is small enough that nutrients can diffuse to all thecells of the DMO from a nutrient medium which contacts the DMO and thatwaste products can diffuse out of the DMO and into the medium. Thisenables the DMO to be viable in vitro long enough for the furtherprocessing described below and for the optional further utilization ofthe DMO as a source for a therapeutic agent, such as a protein. Themethod of harvesting a DMO as described above, generally results in aDMO having an in vitro life of several months.

After the DMO is harvested; it is optionally visually inspected todetermine that it is properly formed and that it has the desireddimensions. Inspection can also be performed optically. It is thenoptionally mounted on a holder and transported (block 206) to anapparatus (the bioreactor, as will be described below) in which it canbe genetically altered. A suitable genetic modification agent isprepared (block 208). Alternative exemplary methods of preparing theagent include creation of aliquots with a desired amount, using apredefined dilution buffer of modifying agent such as for example aviral vector, possible cryogenic storage and thawing of the modifyingagent, under to controlled temperature (0-4.degree. C.), and validatingthe activity of the modifying agent. All of these processes are wellknown in the art. At this point the DMO can be stored cryogenically, forlater introduction at the same place in the process. This can beperformed using known protocols for gradual freezing of tissues andcells, using for example, DMEM medium containing 10% DMSO.

At block 210 the DMO is genetically altered. As described above, manymethods of genetic alteration are known and may be used in conjunctionwith the present invention. As an example, the following description isbased on using a viral vector to insert a gene into the cells of theDMO. This process is well known and will not be further described,except as to the particular methodology and apparatus for introducingthe virus to the DMO.

At block 212 the genetically altered DTMO is optionally tested forproduction and secretion rates of the therapeutic agent. There arevarious methods of determining the quantity of secretion, for example,ELISA, other immunoassays, spectral analysis, etc. In addition thequality of the secretion is optionally tested, for example for sterilityand activity of the secreted protein. This may be performed periodicallyor continuously on-line. At this point the DTMO can be cryogenicallystored for later use.

At blocks 214 and 216, the amount of DTMO required for producing adesired therapeutic effect is determined. As indicated below, thetherapeutic dose requirements can be estimated from measured secretionrates, patient parameters and population statistics on the estimated orknown relationship between in vitro secretion and in vivo serum levels.

At block 218 the selected number of the DTMOs are loaded intoimplantation tools. Exemplary implementation tools have been describedabove. If needed, for allografts or xenografts or for other reasons, theDTMO can be encapsulated. If the DTMO must be transported prior to beingtransported to the implantation tools, it is optionally held (220) in amaintenance station, in which the temperature, humidity, etc. are heldat levels that allow the DTMO to stay viable during transport. Theremaining DTMO material is optionally maintained in vitro for futureuse. This can be at warm incubator conditions (30-37.degree. C.), inconditions as described above or at cool incubator conditions (4.degree.C.), which may prolong its viability in vitro.

At block 224, a subset of the DTMOs is implanted into the subject. Anexemplary embodiment of a method of implantation is described above.Other methods of doing so will occur to persons of skill in the art andare primarily dependent on the specific geometry of the micro-organbeing used. Animal studies have shown that the DMOs and DTMOs remainviable in vivo, in the sense that the DTMO continues to produce andsecrete the therapeutic agent for a period of weeks and months followingimplantation (FIG. 7). In animal studies, therapeutic amounts areproduced for periods up to 160 days (or longer). While the tissue of theDMO or DTMO appears to be integrated or well taken into the tissue ofthe subject into which it is implanted (especially if the tissue isimplanted in a tissue of the same kind from which it was harvested), thecells including the DMO or the DTMO continue to produce and secrete thetherapeutic agent.

In either case, the in vivo performance of the DTMO is optionallydetermined (block 228). Based on this evaluation for example, and/or onpast patient data (block 226), patient dosage may then be adjusted(block 230) by increasing the amount of the implant or removing some ofthe implant, as described below. As the efficacy of the implant changes,additional DTMO can be implanted.

Genetic alteration may generally include genetically engineering aselected gene or genes into cells that causes the cells to produce andoptionally to secrete a desired therapeutic agent such as a protein. Inan embodiment of the invention, at least part of the process ofsustaining the DMO during the genetic alteration, as well as the geneticalteration itself, may be performed in a bioreactor, as described below.

Reference is now made to FIG. 10, which schematically illustrates aflowchart of a method of harvesting a dermal micro-organ according tosome exemplary embodiments of the invention, and to FIGS. 11 a-11 c,which schematically illustrate exemplary stages of harvesting a dermalmicro-organ 1160 located under a skin tissue portion 1120 in accordancewith the method of FIG. 10.

As indicated at block 1002, the method may optionally include locallyadministering an anesthetic, e.g., as is known in the art, to thevicinity of the DMO to be harvested.

As indicated at block 1004, the method may further include inserting aninner guide 1110 into tissue portion 1120. Thin incisions (“lance cuts”)1190 and 1130 may be formed in the outer skin, preferably using asurgical lance, scalpel, or other sharp probe, in order to allow easierinsertion of inner guide 1110, and also to prevent or minimize theharvesting of epidermal tissue. Inner guide 1110 may be inserted intoportion 1120 via incision 1190, e.g., generally parallel to the skinsurface and/or at a desired depth within the dermis or just under theskin. Inner guide 1110 may include a thin needle, rod, or any othersuitable thin, generally straight, object able to be placed inside thedermis or in a subcutaneous space. For example, inner guide 1110 mayinclude a needle of size 20-25 G, for example, about 22 G, as is knownin the art. Inner guide 1110 may be inserted into the dermis orsubcutaneous space and/or pushed generally horizontally, i.e., generallyin parallel with the skin surface. The length of penetration of guide1110 within the dermis may generally correspond to the length of the DMOto be harvested. For example, inner guide 1110 may be inserted manually,and hand guided within the dermis at a desired depth, which depth may bemaintained substantially uniformly throughout the insertion process.Alternatively, inner guide 1110 may be inserted into and along thesubcutaneous space, by manually sensing the boundary between the fibrousdermis and an underlying smooth fatty layer as the inner guide isinserted.

As indicated at block 1006, the method may optionally include guidinginner guide 1110 to exit the skin, e.g., at incision 1130. According tosome exemplary embodiments, the distance between incisions 1190 and 1130may be approximately equal to or larger than a required length of theDMO to be harvested.

As indicated at block 1008, the method may also include inserting atubular cutting tool coaxially with and around inner guide 1110, suchthat the DMO may be trapped, i.e., positioned, between the inner guide1110 and the cutting tool. This may be achieved, for example, by using atubular cutting tool having an inner diameter larger than the outerdiameter of inner guide 1110. The cutting tool may include any suitablecutting tool, for example, a coring tube 1150. Coring tube 1150 mayinclude a generally symmetrically sharpened tubular tool, e.g., a hypotube processed to have sharpened cutting edge with a desired shape.Coring tube 1150 may include, for example, a standard medical gradetube, having a thin wall, e.g., having a thickness of between 0.05 mmand 0.3 mm Coring tube 1150 may have a diameter, for example, between 1mm and 10 mm. The dimensions, e.g., the diameter, of coring tube 1150and/or the dimensions of inner guide 1110 may be predetermined based onthe volume and/or dimensions of the DMO intended to be harvested. Coringtube 1150 may have a sharpened end (“tip”) 1140 adapted to serve as acutting edge. Coring tube 1150 may be inserted through tissue portion1120, preferably after creating initial incisions, E.G., INCISION 1130,on the outer surface of the skin in order to prevent harvesting ofepidermal tissue.

According to one exemplary embodiment of the invention, e.g., asillustrated in FIG. 11 b, the method may include initially positioningend 1140 of coring tube 1150 over a distal end of inner guide 1110,e.g., at incision 1130, and sliding coring tube 1150 along the length ofinner guide 1110, e.g., towards incision 1190, to harvest the dermalDMO.

As indicated at block 1010, in one embodiment the method may includerotating the cutting tool while advancing the cutting tool, e.g.,towards the proximal end of the inner guide. For example, a medicaldrill or other suitable tool or rotation mechanism may be used to rotatecoring tube 1150 while it is advanced manually or automatically, therebymore smoothly harvesting DMO 1160. For example, a proximal end 1180 ofcoring tube 1150 may be connected to a medical drill 1170, such as, forexample, the Aesculap Micro Speed drill manufactured by Aesculap AG &Co. KG, Am Aesculap Platz, D-78532 Tuttlingen, Germany, which mayinclude a control unit, a motor, a connection cord, a hand piece and/ora foot switch, catalogue numbers GD650, GD658, GB661, GB166 and GB660,respectively. Such a drill, or any other suitable drill or rotationmechanism, may be used to rotate the cutting edge of the cutting tool ata rotational speed appropriate for cutting of the dermal tissue, forexample, a relatively high rotational speed, for example, a speed higherthan 1,000 RPM, e.g., between 1,000 RPM and 10,000 RPM. For example,tube 1150 may be rotated at a rotational speed higher than 2,000 RPM,e.g., approximately 7,000 RPM. Alternatively, a relatively lowrotational speed of less than 1000 RPM may be used, or no rotation atall, as described below. Optionally, the rotational speed of the drillmay vary in an oscillatory manner, i.e., the direction of rotation mayvary periodically between “clockwise” and “counterclockwise” directions.While rotated by drill 1170, coring tube 1150 may be manually orautomatically advanced, e.g., towards the proximal end of inner guide1110, e.g., towards incision 1190. The method may also include stoppingthe forward motion of coring tube 1150, for example, when tip 1140 hasbeen advanced just beyond incision 1190. According to some exemplaryembodiments of the invention, at least part of an inner surface and/oran outer surface of tube 1150 may be coated with a low frictionmaterial, e.g., Teflon, Parylene or any other suitable coating material,e.g., to ease the separation of the harvested tissue from the innersurface of the cutting tool in a subsequent action and/or to reduce anyforces acting on the tissue during the cutting action, as describedbelow.

In another embodiment, a fast-acting, e.g., spring-loaded, insertionmechanism may be used to assist coring tube 1150 in penetrating theharvesting target and cutting the dermis, e.g., with substantially norotational motion of the coring tube.

As indicated at block 1012, the method may include withdrawing innerguide 1110, e.g., having DMO 1160 impaled thereon, from within coringtube 1150, thereby to extract DMO 1160 from portion 1120.

According to some embodiments, DMO 1160 may be left impaled on innerguide 1110. In to such a case, inner guide 1110 may be used to handle,transport, and/or manipulate the DMO 1160. Alternatively DMO 1160 maybe, for example, carefully removed from inner guide 1160 into abioreactor processing chamber, e.g., as described in detail below withreference to FIG. 22, or onto various transfer devices (not shown)adapted for transferring the DMO to a different mount or into a chamberfor further processing. Such transfer devices may include, for example,forceps, vacuum grippers or any other mechanical devices able to gripDMO 1160 and/or push DMO 1160 off inner guide 1110. In addition,suitable fluids, such as sterile fluids, may be used, either alone or inconjunction with the means listed above, to assist in removing the DMOfrom inner guide 1160.

As indicated at block 1014, the method may also include withdrawing thecutting tool, e.g., coring tube 1150, from skin portion 1120.

It will be appreciated by those skilled in the art that any combinationof the above actions may be implemented to perform harvesting accordingto embodiments of the invention. Further, other actions or series ofactions may be used.

According to some embodiments of the invention, the harvesting methodmay additionally include externally stabilizing and/or supporting theDMO to be harvested and/or tissue in the vicinity of the DMO to beharvested e.g., using an external support device and/or mechanism, forexample, in addition to internally stabilizing and/or supporting thedermis, e.g., by the inner guide, as described below.

Reference is also made to FIG. 12, which schematically illustrates astabilizing clamping tool 1200, which may be used in conjunction with adermal harvesting apparatus in accordance with some exemplaryembodiments of the invention.

According to exemplary embodiments of the invention, tool 1200 mayinclude a clamping mechanism having clamping edges 1210. For example,tool 1200 may include a pinching clamp or forceps, e.g., as are known inthe art. Tool 1200 may include a spring clamp having a constant clampingforce, or a controllably variable clamping force. Tool 1200 may beplaced on the skin surface parallel to and on either side of inner guide1110, e.g., such that when closed, clamping edges 1210 may be positionedbeneath inner guide 1110. Clamping edges 1210, when brought closetogether, may function to stabilize and/or support inner guide 1110and/or a skin portion 1240 associated with the DMO to be harvested, suchthat the DMO may be stabilized while being cut by tube 1150. Coring tube1150, in this case, may be pushed through clamping edges 1210 concentricor non-concentric to inner guide 1110, while force is applied. Accordingto some exemplary embodiments of the invention, clamping edges 1210 mayinclude at least one or two rows of serrated teeth 1260 in order toprovide improved clamping of portion 1240 and reduce, e.g., minimize,lateral movement of the skin during the coring process.

Other tools and/or mechanisms may be used to apply force to the outerskin in order to cause similar compression of the dermis surrounding theinner guide. Alternatively, other devices and/or methods for stabilizingthe dermis to be harvested may be used, such as twisting the inner guideand holding it at a substantially fixed position with respect to therotation of the coring tube.

Reference is also made to FIG. 13, which schematically illustrates across sectional view of coring tube 1150 inserted coaxially over andalong inner guide 1110 in accordance with some exemplary embodiments ofthe invention.

According to some embodiments of the present invention, inner guide 1110may be placed in skin portion 1120 at a position such that an axis 1125of guide 1110 is positioned substantially at the center of DMO 1160. Insuch a case, coring tube 1150 may be substantially coaxially alignedwith inner guide 1110, such that DMO 1160 is impaled on inner guide 1110in an approximately symmetrical manner.

However, according to other exemplary embodiments of the invention, theinner guide and the coring tube may be positioned in any other suitablearrangement. For example, the inner guide may be positioned in thesubcutaneous space, such that the desired DMO to be harvested may beprimarily located above the inner guide and wrapped around it.Accordingly, the coring tube may be inserted over the inner guide and/orguided such that the inner guide is positioned close to or touches thelower inner surface of the coring tube as it cuts the DMO. In such acase, the inner guide may hold the DMO, which may rest, for example,along the upper surface of the inner guide when being removed.

According to some embodiments of the present invention, the abovedescribed manual procedures may be facilitated by an integratedapparatus (not shown) configured to perform some or all of the aboveprocedures for harvesting the DMO. For example, in regard to oneharvesting method embodiment, the integrated apparatus may be configuredto enable positioning and guiding the insertion of inner guide 1110,attaching clamping tool 1200, guiding the insertion of coring tube 1150and controlling its movement during the cutting process, and/or removingDMO 1160 being attached to inner guide 1110. Such an apparatus mayenable relatively simple operation when performing a harvestingprocedure.

According to some exemplary embodiments of the invention, a method ofharvesting a DMO from a subject may include generating and/ormaintaining a skin-related tissue structure associated with the DMO,e.g., located generally at a targeted harvest site for harvesting theDMO, at a desired shape and position such that the cutting tool may beable to separate at least part of the DMO from tissue in the vicinity ofthe DMO. For example, an epidermis portion in the vicinity of thetargeted harvest site may be lifted, e.g., by attaching at least part ofthe epidermis portion to a predefined, e.g., substantially flat, surfacearea such that at least part of the skin-related tissue structure may belifted and maintained at the desired shape and/or position. According tosome exemplary embodiments, attaching the epidermis to the predefinedsurface may include applying a vacuum condition, e.g., as describedbelow. Alternatively or additionally, attaching the epidermis to thepredefined surface may include applying an adhesive to the surface.

Reference is now made to FIGS. 14 a-14 c, which schematically illustratea front view, a side view, and a top view, respectively, of a dermalharvesting apparatus 1400 for harvesting a DMO according to oneexemplary embodiment of the invention, and to FIG. 15, whichschematically illustrates a cross-section side view of apparatus 1400being implemented for externally supporting a skin-related tissuestructure including DMO 1510 at a desired position according to oneexemplary embodiment of the invention.

Apparatus 1400 may include a vacuum chamber, e.g., a generallycylindrical longitudinal chamber 1406, having a top support surface 1430fluidically connected via a plurality of channels 1404 to a vacuum inlet1402. Vacuum inlet 1402 may be fluidically connected to at least onvacuum source, e.g., a vacuum pump (not shown), to provide a vacuumcondition to chamber 1406. Surface 1430 and/or channels 1404 may beconfigured to enable attaching to surface 1430 at least part of anepidermal layer 1508 associated with DMO 1510, e.g., located generallyabove DMO 1510, when a vacuum condition is applied to chamber 1406,e.g., by the vacuum source.

Apparatus 1400 may also include a guiding channel 1416 for guiding acutting tool, e.g., a coring tube 1520, and maintaining the cutting toolat a predetermined location, e.g. a predetermined to distance from uppersurface 1430. For example, the upper surface of cutting tool 1520 may belocated at a distance, for example, of approximately 1 mm from uppersurface 1430. In other embodiment, other ranges, such as for example,0.3-2.0 mm, may also be used. Channel 1416 may include, for example, agenerally cylindrical channel having a diameter slightly larger than theouter diameter of coring tube 1520. Coring tube 1520 may include acoring needle having a size of, e.g., between 1 mm and 10 mm, forexample, 14 G (corresponding to an outer diameter of approximately 2.11mm) and having a symmetrically sharpened cutting edge.

According to exemplary embodiments of the invention, surface 1430 may beflat, generally curved, or may have any other suitable shape. Forexample, in one embodiment, surface 1430 may have a radius of curvatureof about 3.5 mm. In one embodiment, chamber 1406 may have a width of,for example, about 4 mm. Furthermore, in some embodiments, chamber 1406may have a height of, for example, about 5 mm. In other embodiments,other ranges, such as for example, 3-25 mm, may also be used for theradius of curvature of surface 1430 and/or the width and/or height ofchamber 1406, for example, any desired dimensions in the range of 3-25mm may be used in some embodiments of the invention. The length ofchamber 1406 may be generally similar to the length of the DMO beingharvested, for example, approximately 30 mm in length; however, otherranges, for example, in the range of 5-100 mm, may be used for thechamber length.

According to some exemplary embodiments, apparatus 1400 may include twochannels 1408 located at least partially along two sides of chamber1406, respectively, to allow clamping epidermis layer 1508, as describedbelow. Channels 1408 may be positioned, e.g., centered, at a desiredheight, for example, at approximately the same height as where thecenter of the DMO is to be harvested. In one embodiment, the center ofchannels 1408 may be positioned at a height of about 2 mm below uppersurface 1430. so that the clamping may stabilize and/or support thetissue being cut. According to these exemplary embodiments, apparatus1400 may also include two flexible membrane elements 1412, on either theinner surface or outer surface of channels 1408, so as to allow externalclamping of the tissue without substantially affecting the vacuumcondition applied to chamber 1406. According to other embodiments of theinvention, apparatus 1400 may not include elements 1412 and/or channels1408.

According to exemplary embodiments of the invention, a method ofharvesting DMO 1510 using apparatus 1400 may include forming twoincisions (not shown), e.g., forming two lance cuts using a scalpel, ina skin portion associated with DMO 1510 at a predetermined distance,e.g., approximately 30 mm, which may correspond to the points at whichcoring tube 1520 is intended to enter and exit epidermis 1508 (“theentry and exit penetration sites”). The incisions may be formed in orderto ensure that there will be substantially no epidermal component at thetwo ends of harvested DMO 1510, and/or to maintain a desired shape ofthe penetration sites such that they may heal efficiently, i.e., quicklyand/or leaving relatively small scars. The method may also includeplacing apparatus 1400 in contact with epidermis layer 1508 (“theharvest site”) such that the incisions are positioned underneath chamber1406, i.e., in between points 1410 and 1414. The incisions may bepositioned at points 1410 and/or 1414, respectively, or may bepositioned between points 1410 and 1414 to help force the lance cuts to“open” once the vacuum condition is applied to chamber 1406. Accordingto some exemplary embodiments, apparatus 1400 may optionally include amechanism configured for creating the lance cuts, for example, springloaded lancets that produce the lance cuts, e.g., after apparatus 1400is placed on the harvest site and before the vacuum condition is appliedto chamber 1406.

The method may also include inserting coring tube 1520 into channel1416. Coring tube 1520 may be connected, for example, via a connector,e.g., a Jacobs Chuck or a friction holder, to a medical drill or anyother suitable tool and/or mechanism, e.g., drill 1170 (FIG. 11), ableto rotate coring tube 1520. Optionally, the rotational speed of thedrill may vary in an oscillatory manner, i.e., the direction of rotationmay vary periodically between “clockwise” and “counterclockwise”directions.

The method may also include applying a vacuum condition to chamber 1406,e.g., by activating the vacuum source. Consequently, the skin-relatedtissue structure may be drawn into chamber 1406 and epidermis 1508,e.g., between the lance cuts, may be firmly held against surface 1430.Epidermis 1508, dermis 1506, and/or fatty tissue components 1504 mayadditionally be drawn into chamber 1406, depending on the thickness ofeach of these tissue layers and the dimensions of chamber 1406. Thus,the dimensions of chamber 1406 may be designed in accordance with theanticipated thickness of one or more of the tissue layers and/orexterior clamping, e.g., as described herein, may be applied such thatfat tissue 1504 drawn into vacuum chamber 1406 may be forced downwardsand substantially out of chamber 1406.

The method may further include rotating coring tube 1520, e.g., usingdrill 1170 (FIG. 11) at a relatively high rotational speed, e.g., higherthan 1,000 RPM, e.g., between 1,000 RPM and 10,000 RPM. For example,coring tube 1520 may be rotated at a rotational speed higher than 2,000RPM, e.g., approximately 7,000 RPM. Alternatively, a relatively lowrotational speed of less than 1000 RPM may to be used, or no rotation atall, as described above. The method may also include advancing coringtube 1520 along vacuum chamber 1406, e.g., at least along the entirelength of chamber 1406. Coring tube 1520 may be guided through channel1416 in order to ensure that dermal micro-organ 1510 is harvested fromapproximately the same depth in the skin-related tissue structure alongchamber 1406. Coring tube 1520 may be advanced manually, or using amotorized actuator (not shown), e.g., to control the speed at whichcoring tube 1520 may advance.

The method may also include detaching DMO 1510 from tissue surroundingDMO 1510. For example, apparatus 1400 may include an extension 1418,e.g., having a length of between 1 mm and 5 mm and a radiussubstantially equal to the radius of channel 1416, located substantiallyopposite channel 1416 such that coring tube 1520 may advance intoextension 1418 after going through chamber 1406. Alternatively, acutting surface 1440, e.g., formed of Silicone or other suitablematerial, may be positioned in extension 1418 such that the coring tubemay, cut into surface 1440 to detach the harvested DMO. Additionally, avacuum condition may be applied within coring tube 1520, e.g., from itsback end, such that DMO 1510 may be actively drawn into coring tube1520, thus urging final detachment of the DMO from the surroundingtissue.

The method may further include withdrawing coring tube 1520, includingtherein DMO 1510, from apparatus 1400.

Reference is made to FIG. 16, which schematically illustrates across-sectional side view of apparatus 1400 being implemented forexternally supporting a skin-related tissue structure at a desiredposition according to another exemplary embodiment of the invention.

According to the exemplary embodiment of FIG. 16, improved stabilizationof dermis 1506 and/or improved prevention of recruitment of fat 1504into vacuum chamber 1406 may be accomplished by external clamping of theskin-related tissue structure supported within the vacuum chamber. Forexample, a clamping tool 1600, e.g., analogous to the clamping tooldescribed above with reference to FIG. 12, may be implemented to “pinch”the skin-related tissue structure supported inside vacuum chamber 1406,e.g., symmetrically. Two clamping ends 1502 of clamping tool 1600 may beinserted into channels 1408, respectively. Tool 1600 may be closed suchthat clamping ends 1502 may press down against flexible elements 1412.Thus, the skin-related tissue structure in chamber 1406 may be clampedfrom the sides without substantially affecting the vacuum condition inchamber 1406. A clamping force applied by clamping ends 1502 maycorrespond, for example, to a constant or variable force of a spring1512 or other suitable device.

Although the above description may refer to a vacuum chamber having agenerally constant shape and/or size along its longitudinal axis, itwill be appreciated by those skilled in the art that, according to otherembodiments of the invention, the vacuum chamber may have any otherpredetermined size and/or shape, e.g., as described below.

Reference is now made to FIG. 17, which schematically illustrates adermal harvesting apparatus 1700 according to another exemplaryembodiment of the invention.

Apparatus 1700 may include a vacuum chamber 1701 including an elevatedprotrusion 1706. Elevated protrusion 1706 may have a predetermined sizeand/or shape adapted, for example, to enable the creation of a “plateau”of a single layer of skin tissue in a generally flat orientation,elevated above the trajectory of a coring tube 1716. For example,section 1706 may be higher than other sections of chamber 1701, suchthat a fat layer 1718 may be drawn into section 1706 and supported alongthe trajectory of coring tube 1716. As a result, after harvesting a DMOof a predetermined length, coring tube 1716 may be slightly advancedinto fat layer 1718, thus separating the harvested DMO from tissuesurrounding the DMO. The harvested DMO may remain within coring tube1716 as it is withdrawn from the body. The configuration of Apparatus1700 may eliminate the need for forming an “exit” incision in the skin,e.g., as described above, thus enabling the harvesting of a DMO withonly a single incision.

According to some exemplary embodiments of the invention, apparatus 1700may also include a drill stopper 1708 to enable manually advancingcoring tube 1716 for a predetermined distance along chamber 1701, e.g.,to a position in which coring tube 1716 has slightly advanced into fattissue 1718.

Reference is now made to FIG. 18, which schematically illustrates aharvesting apparatus 1800, according to yet another exemplary embodimentof the invention, and to FIG. 19, which schematically illustrates across sectional view of apparatus 1800 being implemented for harvestinga DMO 1830.

According to some exemplary embodiments, core biopsy devices withsimilarities to the devices used, for example, in breast cancer biopsyapplications, as described below, may be utilized for harvesting a DMO.Apparatus 1800 may include a cutting tool 1808, e.g., as describedabove, and a to Subcutaneous Harvest Trocar (HST) 1806, e.g., ahypodermic needle with a sharpened tip 1804 and a suitable innerdiameter, e.g., being slightly larger than the outer diameter of cuttingtool 1808, such that cutting tool 1808 may be inserted into andsubstantially coaxially within HST 1806. HST 1806 may include a notchcutout (“window”) 1802 of a suitable depth, e.g., 1 mm or more, and asuitable length, e.g., substantially equal to the desired length of theDMO to be harvested.

According to the exemplary embodiments of FIG. 18, a single incision,e.g., lance cut, may be formed, e.g., using a scalpel blade, throughwhich HST 1806 may be inserted together with cutting tool 1808, e.g., asa single unit, at the desired position underneath or in the skin,preferably in the subcutaneous space with notch 1802 oriented upwardstowards dermis layer 1840. Cutting tool 1801 may be positioned withinHST 1806 during penetration such that window cutout 1802 may be “closed”to allow a generally smooth penetration of HST 1806. Tool 1808 and HST1806 inserted therein may run along the subcutaneous interface for thelength of notch 1802, and end 1804 may not exit through the skinsurface. Once appropriately positioned, tool 1808 may be retracted toexpose notch 1802 and allow for dermal tissue to substantially fill thenotch. Appropriate pressure on the skin surface may be applied, e.g.,using a suitable clamping tool, for example, as described above withreference to FIG. 12, and/or a vacuum condition may be applied fromwithin HST 1802 by a vacuum manifold (not shown), e.g., located undernotch cutout 1802, to assist the dermis to substantially fill notch1802. Tool 1808 may be connected to a motor, e.g., as described above,to rotate tool 1808 at a rotational speed appropriate for cutting of thedermal tissue, for example, a relatively high rotational speed, forexample, a speed higher than 1,000 RPM, e.g., between 1,000 RPM and10,000 RPM. For example, tool 1808 may be rotated at a rotational speedhigher than 2,000 RPM, e.g., approximately 7,000 RPM. Tool 1808 may thenbe advanced e.g., manually or automatically, for example, until itpasses beyond the end of window cutout 1802, to cut DMO 1830 withinnotch 1802. When complete, the forward and rotational movements of tool1808 may be stopped, and cutting tool 1808 may be retracted withharvested DMO 1830 within it. SHT 1806 may then be removed from theharvest site. DMO 1830 may be removed from cutting tool 1808, e.g.,using a syringe to flush sterile fluid, for example saline, through tool1808, or a vacuum source to draw out DMO 1830 from a back end (notshown) of cutting tool 1808.

It will be appreciated by those skilled in the art that apparatus 1800may enable harvesting of the DMO by forming only one incision.Furthermore, apparatus 1800 may be efficiently applied for harvesting aDMO from areas having relatively thick skin, e.g., from a region of thedonor's back.

It will be appreciated by those skilled in the art that the harvestingmethods and/or apparatuses according to embodiments of the invention,e.g., as described above, may include introducing thin tissue cuttingdevices within the dermis. Thus, the harvesting methods and/orapparatuses according to embodiments of the invention may enableharvesting the DMO with relatively minimal damage to the outer skinsurface, and therefore may provide a minimally invasive method ofharvesting the desired tissues.

Although some embodiments of the invention described herein may refer tomethods and/or apparatuses for harvesting a DMO, it will be appreciatedby those skilled in the art that according to other embodiments of theinvention at least some of the methods and/or apparatuses may beimplemented for any other procedures, e.g., plastic surgical procedures,dermatological procedures, or any other procedures including harvestingof tissues. For example, the methods and/or apparatuses according toembodiments of the invention may be implemented for harvesting dermaltissue to be used, e.g., in a subsequent implantation, as fillermaterial.

According to some embodiments of the present invention, a system andmethod are provided for ex-vivo (“in vitro”) handling or processing ofdermal micro-organs. Dermal tissue that has been harvested as a directMO may be left on their inner guide as a mount for the MO. In theseembodiments, the inner guide may be used to maintain position andorientation of the MOs during subsequent processing. In otherembodiments, the dermal MOs may be removed from the inner guide anddirectly placed into tissue culture wells or transduction chambers of abioreactor, as described in detail below, e.g., with reference to FIG.22. In some embodiments, e.g., if the DMO remains in the coring tube asit is withdrawn from the skin, the DMO may be flushed out of the coringtube by the use of biologically compatible fluid, e.g., saline or growthmedium, applied to the back end of the coring tube. The flushing of theDMO may be such that it is flushed directly into a chamber of thebioreactor, e.g., as described below. Alternatively, vacuum may beapplied to a back end of the coring tube to “draw out” the DMO, e.g.,directly into a chamber of the bioreactor.

According to some embodiments of the present invention, a system andmethod are provided for implantation of DTMOs. After producing and/orprocessing of a DMO, for example, by genetically modifying the DMO, themodified DMO or DTMO may be implanted back into the patient, forexample, for protein or RNA based therapy. The number of full or partialDTMOs that are implanted may be determined by the desired therapeuticdosing of the secreted protein. DTMOs may be implanted subcutaneously orat any other locations within the body. Subcutaneous implantation by useof a needle trocar, for example, may enable the DTMO to remain in alinear form in the subcutaneous space. The linear form of implantationmay help facilitate localization in case later ablation of the DTMO isrequired, for example, in order to stop treatment or reduce the dose oftherapeutic protein. Other known geometrical implantation patterns couldbe used. The linear implantation may also assist in the integration ofthe dermal tissue to the surrounding tissue.

Reference is now made to FIG. 20, which schematically illustrates aflowchart of a method of implanting a DTMO according to some exemplaryembodiments of the invention.

As indicated at block 2002 a local anesthetic may be optionallyadministered at an intended implantation site.

As indicated at block 2004, according to some exemplary embodiments ofthe invention, the DTMO, optionally together with surrounding sterilesaline fluid may be aspirated into a carrier, for example, animplantation needle, e.g., attached to a syringe. The needle may haveany suitable diameter, for example, between 17-gauge and 12-gauge.Optionally, a tip of the needle may have a short extension of silicontubing, or the like, affixed to it, to facilitate the aspiration of theDTMO into the needle cannula while retracting the plunger of thesyringe.

As indicated at block 2006, with the loaded DTMO, the implantationneedle, may be pushed into the skin, e.g., without the silicon tubingextension, into the subcutaneous destination, along a distanceapproximately equivalent to the length of the DTMO.

As indicated at block 2008, according to some embodiments, theimplantation needle may exit through the skin surface at a distal end ofthe implantation site.

According to some exemplary embodiments of the invention, the method mayinclude applying pressure on the aspirated dermal therapeuticmicro-organ such that the dermal therapeutic micro-organ exits from thecarrier into the implantation site.

As indicated at block 2010, the tip of the DTMO may be grasped at theexit point with a gripping tool, for example tweezers.

As indicated at block 2012, the implantation needle may be retractedthrough the subcutaneous space, releasing the DTMO from the implantationneedle and laying the DTMO linearly to along the needle tract.Assistance may be given to help release the DTMO, if needed, for exampleby gently pushing down on the syringe plunger during retraction.

As indicated at block 2014, once the DTMO has been left in place, thetip of the DTMO may be released by the gripping tool.

According to some embodiments of the present invention, a system andmethod are provided for in-vivo demarcation and localization of theimplanted dermal micro-organs. Identification of the location of asubcutaneous implantation or implantation at any other location in thebody, of processed tissue, such as a DTMO, may be important, forexample, in the case where it is necessary to stop the proteintreatment, or to decrease the dosage of the secreted protein. Forexample, termination or titration of dosage may be performed by removingone or more DTMOs entirely and/or by ablating one, a portion of one, ormore than one of the implanted DTMOs. In order to identify asubcutaneously implanted DTMO, according to one embodiment, the DTMO maybe colored prior to implantation by an inert, biocompatible ink or staincontaining, for example, a chromophore, which may be visible to thenaked eye or may require special illumination conditions to visualizeit. In this way a DTMO may be distinguished from its surrounding tissueby visual inspection and/or by use of enhanced imaging means.

According to one embodiment, the peripheral surface of a DTMO may becoated with, for example, biocompatible carbon particles, biocompatibletattoo ink, or other suitable materials. Once implanted subcutaneously,the DTMO may be visible with the naked eye or with a suitable enhancedimaging device. Other ways to enhance the visibility of an implantedDTMO may include using a strong light source above the skin surface, orpinching the skin and directing the light source at the skin from oneside, such that the skin may appear translucent and the dyed DTMO may bemore visible. Alternatively, the stain may be fluorescent, visible onlywhen illuminated using UV light, such as using fluorescent plasticbeads.

According to another embodiment, the location of a subcutaneouslyimplanted DTMO may be identified by co-implanting a biocompatiblestructure along with the DTMO. An example of such a biocompatiblestructure is a non-absorbable single stranded nylon suture commonly usedin many surgical procedures. Such a suture may be implanted in the sameimplantation tract with the DTMO, or may be implanted directly above theDTMO in the upper dermis, such that the spatial location of the DTMO maybe determined by the suture location. Further, the depth of the DTMO maybe known to be at the depth of the subcutaneous space. The suture may bevisible to the naked eye, observed with the assistance of illuminationmeans, and/or observed with the aid of other suitable imaging means,such as ultrasound. Alternatively, the suture can be fluorescent, andvisible through the skin under appropriate UV illumination. The suturemay alternatively be of an absorbable material, so that it may enabledetermination of localization for a desired period of time, such as afew months.

According to another embodiment, the DTMO may be genetically modified orengineered to include a gene to express a fluorescent marker or othermarker capable of being visualized. For example, the DTMO can bemodified with the GFP (Green Fluorescent Protein) gene or Luciferasereported gene, which, for example, may be expressed along with the genefor the therapeutic protein. In this manner, the DTMO may be visualizednon-invasively using appropriate UV or other suitable illumination andimaging conditions.

According to some embodiments of the present invention, a system andmethod are provided for removal or ablation of implanted DTMOs. In acase, for example, where DTMO-based therapy to a patient must beterminated, or if the protein secretion must be decreased, eachimplanted DTMO may be partially or entirely removed, or partially orentirely ablated. One embodiment for removal of a DTMO is by means of acoring tube similar to, or slightly larger in diameter than, that usedfor direct harvesting of the DMO.

As can be seen with reference to FIG. 21, at block 2102 the location ofthe implanted subcutaneous DTMO may be determined. At block 2103, alocal anesthetic may be optionally administered at the site of DTMOremoval. At block 2104 an inner guide may be inserted subcutaneouslyalong the length of the DTMO, to harvest a core of tissue, whichincludes the DTMO. At block 2106 a coring needle, of the same or largerdiameter than that of the implantation needle (for example, 11-gauge orsimilar), may be inserted concentrically over the inner guide. At block2108 a core of tissue that includes the DTMO may be harvested. At block2110 the inner guide with the cored of tissue and the coring needle maybe extracted from the skin, with the DTMO. In one embodiment, such acoring approach may be combined with vacuum suction to help remove thecut material from the body.

According to an embodiment of the present invention, minimally invasiveor non-invasive methods of ablating the DTMO in-situ may be used to makethe procedure less traumatic and less invasive for the patient. In oneembodiment, in the case of the dyed DTMO, a laser, for example, anon-invasive Yag laser may be used. The energy of the Yag laser, forexample, may be selectively absorbed by the chromophore, such that theenergy is primarily directed to the DTMO, with minimum damage caused tothe surrounding tissue. Other light energy sources may also be used.

According to another embodiment, the DTMO may be ablated by deliveringdestructive energy from a minimally invasive probe inserted into thesubcutaneous space along the length of the DTMO. Such a probe may enabledelivery of a variety of energy types, including radio frequency,cryogenic, microwave, resistive heat, etc. A co-implanted structure,such as a suture, may be used to determine the location of the DTMO,thereby enabling the probe to be inserted subcutaneously, for example,along or directly below the suture. In such a case, for example, thedestructive energy may be delivered while the suture is still in place.Alternatively, the suture may be removed after placement of the probeand before delivery of the destructive energy. The amount of energyapplied may be either that required to denature the proteins in thetissue such as during coagulation by diathermy. Additionally oralternatively, the amount of energy applied may be as much as is used inelectro-surgical cutting devices, which char tissue. Of course, othermeans of localization and other means of delivering destructive energymay be used.

After a DMO is harvested, e.g., according to embodiments of the presentinvention, the DMO is optionally genetically altered. Any methodologyknown in the art can be used for genetically altering the tissue. Oneexemplary method is to insert a gene into the cells of the tissue with arecombinant viral vector. Any one of a number of different vectors canbe used, such as viral vectors, plasmid vectors, linear DNA, etc., asknown in the art, to introduce an exogenous nucleic acid fragmentencoding for a therapeutic agent into target cells and/or tissue. Thesevectors can be inserted, for example, using any of infection,transduction, transfection, calcium-phosphate mediated transfection,DEAE-dextran mediated transfection, electroporation, liposome-mediatedtransfection, biolistic gene delivery, liposomal gene delivery usingfusogenic and anionic liposomes (which are an alternative to the use ofcationic liposomes), direct injection, receptor-mediated uptake,magnetoporation, ultrasound and others as known in the art. This geneinsertion is accomplished by introducing the vector into the vicinity ofthe DMO so that the vector can react with the cells of the DMO. Once theexogenous nucleic acid fragment has been incorporated into the cells,the production and/or the secretion rate of the therapeutic agentencoded by the nucleic acid fragment can be quantified.

According to some exemplary embodiments of the invention, the geneticmodification of the DMO may modify the expression profile of anendogenous gene. This may be achieved, for example, by introducing anenhancer, or a repressible or inducible regulatory element forcontrolling the expression of the endogenous gene.

In another embodiment, the invention provides a method of delivering agene product of interest into a subject by implanting the geneticallymodified DMO of the invention into a subject.

As indicated above, the DMO may be in contact with a nutrient solutionduring the process. Thus, a therapeutic agent generated by the DTMO maybe secreted into the solution where its concentration can be measured.The gene of interest may be any gene which encodes to any RNA molecule(sense or antisense), peptide, polypeptide, glycoprotein, lipoprotein orcombination thereof or to any other post modified polypeptide. In oneembodiment of the invention, the gene of interest may be naturallyexpressed in the tissue sample. In another embodiment of this invention,the tissue sample may be genetically engineered so that at least onecell will express the gene of interest, which is either not naturallyexpressed by the cell or has an altered expression profile within thecell.

As used herein, the term “nucleic acid” refers to polynucleotide or tooligonucleotides such as deoxyribonucleic acid (DNA), and, whereappropriate, ribonucleic acid (RNA) or mimetic thereof. The term shouldalso be understood to include, as equivalents, analogs of either RNA orDNA made from nucleotide analogs, and, as applicable to the embodimentbeing described, single (sense or antisense) and double-strandedpolynucleotide. This term includes oligonucleotides composed ofnaturally occurring nucleobases, sugars and covalent internucleoside(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

As is known to those of skill in the art, the term “protein”, “peptide”or “polypeptide” means a linear polymer of amino acids joined in aspecific sequence by peptide bonds. As used herein, the term “aminoacid” refers to either the D or L stereoisomer form of the amino acid,unless otherwise specifically designated. Also encompassed within thescope of this invention are equivalent proteins or equivalent peptides,e.g., having the biological activity of purified wild type tumorsuppressor protein. “Equivalent proteins” and “equivalent polypeptides”refer to compounds that depart from the linear to sequence of thenaturally occurring proteins or polypeptides, but which have amino acidsubstitutions that do not change it's biologically activity. Theseequivalents can differ from the native sequences by the replacement ofone or more amino acids with related amino acids, for example, similarlycharged amino acids, or the substitution or modification of side chainsor functional groups.

The protein, peptide, polypeptide glycoprotein or lipoprotein can be,without being limited, any of the following proteins or variouscombinations thereof: protease, a lipase, a ribonuclease, adeoxyribonuclease, a blood clotting factor, a cytochrome p450 enzyme, atranscription factor, a MHC component, a cytokine, an interleukin, aBMP, a chemokine, a growth factor, a hormone, an enzyme, a monoclonalantibody, a single chain antibody, an oxidoreductas, a p450, aperoxydase, a hydrogenase, a dehydrogenas, a catalase, a transferase, ahydrolase, an isomerase, a ligase, an aminoacyl-trna synthetase, akinase, a phosphoprotein, a mutator transposon, an oxidoreductas, acholinesterase, a glucoamylase, a glycosyl hydrolase, atranscarbamylase, a nuclease, a meganuclease, a ribonuclease, an atpase,a peptidase, a cyclic nucleotide synthetase, a phosphodiesterase, aphosphoprotein, a DNA or RNA associated protein, a high mobility groupprotein, a paired box protein, a histone, a polymerase, a DNA repairprotein, a ribosomal protein, an electron transport protein, a globin, ametallothionein, a membrane transport protein, a structural protein, areceptor, a cell surface receptor, a nuclear receptor, a G-protein, anolfactory receptor, an ion channel receptor, a channel, a tyrosinekinase receptor, a cell adhesion molecule or receptor, a photoreceptor,an active peptide, a protease inhibitor, a chaperone, a chaperonin, astress associated protein, a transcription factor and a chimericprotein.

In one embodiment the amount of protein secreted by the DMO of theinvention is at least 1.6 .mu.g/DTMO/day at the pre-implantation day.

In one embodiment of this invention, the gene of interest may encode toerythropoietin or to equivalent protein thereof.

In one embodiment of this invention, the gene of interest may encode ablood clotting factor or an equivalent protein thereof.

In one embodiment of this invention, the gene of interest may encodeFactor VIII or an equivalent protein thereof.

In one embodiment of this invention, the gene of interest may encodeFactor IX or an equivalent protein thereof.

In another embodiment of the invention, the gene of interest may encode,without limitation, to any of the following proteins, any combination ofthe following proteins and any equivalents thereof: insulin,trypsinogen, chymotrypsinogen, elastase, amylase, serum thymic factor,thymic humoral factor, thymopoietin, gastrin, secretin, somatostatin,substance P, growth hormone, a somatomedin, a colony stimulating factor,erythropoietin, epidermal growth factor, hepatic erythropoietic factor(hepatopoietin), a liver-cell growth factor, an interleukin, a negativegrowth factor, fibroblast growth factor and transforming growth factorof the .beta. family, Interferon .alpha., Interferon .beta.Interferon.gamma., human growth hormone, G-CSF, GM-CSF, TNF-receptor, PDGF, AAT,VEGF, Super oxide dismutase, Interleukin, TGF-.beta., NGF, CTNF, PEDF,NMDA, AAT, Actin, Activin beta-A, Activin beta-B, Activin beta-C Activinbeta-E Adenosine Deaminase adenosine deaminase Agarase-Beta, Albumin HASAlbumin, Alcohol Dehydrogenase Aldolase, Alfimeprase Alpha 1-AntitrypsinAlpha Galactosidase Alpha-1-acid Glycoprotein (AGP),Alpha-1-Antichymotrypsin, Alpha-1Antitrypsin AT, Alpha-1-microglobulinAIM, Alpha-2-Macroglobulin A2M, Alpha-Fetoprotein, Alpha-Galactosidase,Amino Acid Oxidase, D-, Amino Acid Oxidase, L-, Amylase, Alpha, Amylase,Beta, Angiostatin, Angiotensin, Converting Enzyme, Ankyrin,Apolipoprotein, APO-SAA, Arginase, Asparaginase, AspartylAminotransferase, Atrial Natriuretic factor (Anf), Atrial NatriureticPeptide, Atrial natriuretic peptide (Anp), Avidin, Beta-2-Glycoprotein1, Beta-2-microglobulin, Beta-N-Acetylglucosaminidase B-NAG, betaamyloid, Brain natriuretic protein (Bnp), Brain-derived neurotrophicfactor (BDNF), Cadherin E, Calc a, Calc b, Calcitonin, Calcyclin,Caldesmon, Calgizzarin, Calgranulin A, Calgranulin C, Calmodulin,Calreticulin, Calvasculin, Carbonic Anhydrase, Carboxypeptidase,Carboxypeptidase A, Carboxypeptidase B, Carboxypeptidase Y, CARDIACTROPONIN I, CARDIAC TROPONIN T, Casein, Alpha, Catalase, Catenins,Cathepsin D, CD95L, CEA, Cellulase, Centromere Protein B, Ceruloplasmin,Ceruplasmin, cholecystokinin, Cholesterol Esterase, Cholinesterase,Acetyl, Cholinesterase Butyryl, Chorionic Gonadotrophin (HCG), ChorionicGonadotrophin Beta CORE (BchCG), Chymotrypsin, Chymotrypsinogen,Chymotrypsin, Chymotrypsin, Creatin kinase, K-BB, CK-MB (CreatineKinase-MB), CK-MM, Clara cell phospholipid binding protein, Clostripain,Clusterin, CNTF, Collagen, Collagenase, Collagens (type 1-VI), colonystimulating factor, Complement C1q Complement C3, Complement C3a,Complement C3b-alpha, Complement C3b-beta, Complement C4, Complement C5,Complement Factor B, Concanavalin A, Corticoliberin, Corticotrophinreleasing hormone, C-Reactive Protein (CRP), C-type natriuretic peptide(Cnp), Cystatin C, D-Dimer, Delta 1, Delta-like kinase 1 (Dlk1),Deoxyribonuclease, Deoxyribonuclease I, Deoxyribonuclease II,Deoxyribonucleic Acids, Dersalazine, Dextranase, Diaphorase, DNA Ligase,T4, DNA Polymerase I, DNA Polymerase, T4, EGF, Elastase, Elastase,Elastin, Endocrine-gland-derived vascular endothelial growth factor(EG-VEGF), Elastin Endothelin Elastin Endothelin 1 Elastin EotaxinElastin, Epidermal growth factor (EGF), Epithelial Neutrophil ActivatingPeptide-78 (ENA-78), Erythropoietin (Epo), Estriol, Exodus, Factor IX,Factor VIII, Fatty acid-binding protein, Ferritin, fibroblast growthfactor, Fibroblast growth factor 10, Fibroblast growth factor 11,Fibroblast growth factor 12, Fibroblast growth factor 13, Fibroblastgrowth factor 14, Fibroblast growth factor 15, Fibroblast growth factor16, Fibroblast growth factor 17, Fibroblast growth factor 18, Fibroblastgrowth factor 19, Fibroblast growth factor 2, Fibroblast growth factor20, Fibroblast growth factor 3, Fibroblast growth factor 4, Fibroblastgrowth factor 5, Fibroblast growth factor 6, Fibroblast growth factor 7,Fibroblast growth factor 8, Fibroblast growth factor 9, Fibronectin,focal-adhesion kinase (FAK), Follitropin alfa, Galactose Oxidase,Galactosidase, Beta, gamaIP-10, gastrin, GCP, G-CSF, Glial derivedNeurotrophic Factor (GDNF), Glial fibrillary acidic Protein, Glialfilament protein (GFP), glial-derived neurotrophic factor familyreceptor (GFR), globulin, Glucose Oxidase, Glucose-6-PhosphateDehydrogenase, Glucosidase, Alpha, Glucosidase, Beta, Glucuronidase,Beta, Glutamate Decarboxylase, Glyceraldehyde-3-Phosphate Dehydrogenase,Glycerol Dehydrogenase, Glycerol Kinase, Glycogen Phosphorylase ISO BB,Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), growthstimulatory protein (GRO), growth hormone, Growth hormone releasinghormone, Hemopexin, hepatic erythropoietic factor (hepatopoietin),Heregulin alpha, Heregulin beta 1, Heregulin beta 2, Heregulin beta 3,Hexokinase, Histone, Human bone morphogenetic protein, Human relaxin H2,Hyaluronidase, Hydroxysteroid Dehydrogenase, Hypoxia-Inducible Factor-1alpha (HIF-1 Alpha), I-309/TCA-3, IFN alpha, IFN beta, IFN gama, IgA,IgE, IgG, IgM, Insulin, Insulin Like Growth Factor I (IGF-I), InsulinLike Growth Factor II (IGF-II), Interferon, Interferon-inducible T cellalpha chemoattractant (I-TAC), Interleukin, Interleukin 12 beta,Interleukin 18 binding protein, Intestinal trefoil factor, IP10, Jagged1, Jagged 2, Kappa light chain, Keratinocyte Growth Factor (KGF), Kiss1,La/SS-B, Lactate Dehydrogenase, Lactate Dehydrogenase, L-, Lactoferrin,Lactoperoxidase, lambda light chain, Laminin alpha 1, Laminin alpha 2,Laminin beta 1 Laminin beta 2, Laminin beta 3, Laminin gamma 1, Laminingamma 2, LD78beta, Leptin, leucine Aminopeptidase, Leutenizing Hormone(LH), LIF, Lipase, liver-cell growth factor, liver-expressed chemokine(LEC), LKM Antigen,TNF, TNF beta, Luciferase, Lutenizing hormonereleaseing hormone, Lymphocyte activation gene-1 protein (LAG-1),Lymphotactin, Lysozyme, Macrophage Inflammatory Protein 1 alpha (MIP-1Alpha), Macrophage-Derived Chemokine (MDC), Malate Dehydrogenase,Maltase, MCP(macrophage/monocyte chemotactic protein)-1, 2 and 3, 4,M-CSF, MEC (CCL28), Membrane-type frizzled-related protein (Mfrp),Midkine, MIF, MIG (monokine induced by interferon gamma), MIP 2 to 5,MIP-1beta, Mp40; P40 T-cell and mast cell growth factor, Myelin BasicProtein Myeloperoxidase, Myoglobin, Myostatin Growth DifferentiationFactor-8 (GDF-8), Mysoin, Mysoin LC, Mysoin HC, ATPase, NADase, NAP-2,negative growth factor, nerve growth factor (NGF), Neuraminidase,Neuregulin 1, Neuregulin 2, Neuregulin 3, Neuron Specific Enolase,Neuron-Specific Enolase, neurotrophin-3 (NT-3), neurotrophin-4 (NT-4),Neuturin, NGF, NGF-Beta, Nicastrin, Nitrate Reductase, Nitric OxideSynthesases, Nortestosterone, Notch 1, Notch 2, Notch 3, Notch 4, NP-1,NT-1 to 4, NT-3 Tpo, NT-4, Nuclease, Oncostatin M, Ornithinetranscarbamoylase, Osteoprotegerin, Ovalbumin, Oxalate Decarboxylase,P16, Papain, PBP, PBSF, PDGF, PDGF-AA, PDGF-AB, PDGF-BB, PEDF, Pepsin,Peptide YY (PYY), Peroxidase, Persephin, PF-4, P-Glycoprotein,Phosphatase, Acid, Phosphatase, Alkaline, Phosphodiesterase I,Phosphodiesterase II, Phosphoenolpyruvate Carboxylase,Phosphoglucomutase, Phospholipase, Phospholipase A2, Phospholipase A2,Phospholipase C, Phosphotyrosine Kinase, Pituitary adenylate cyclaseactivating polypeptide, Placental Lactogen, Plakoglobin, Plakophilin,Plasma Amine Oxidase, Plasma retinol binding protein, Plasminogen,Pleiotrophin (PTN), PLGF-1, PLGF-2, Pokeweed Antiviral Toxin,Prealbumin, Pregnancy assoc Plasma Protein A, Pregnancy specific beta Iglycoprotein (SPI), Prodynorphin, Proenkephalin, ProgesteroneProinsulin, Prolactin, Pro-melanin-concentrating hormone (Pmch),Pro-opiomelanocortin, proorphanin, Prostate Specific Antigen PSA,Prostatic Acid Phosphatase PAP, Prothrombin, PSA-A1, Pulmonarysurfactant protein A, Pyruvate Kinase, Ranpimase, RANTES, Reelin, Renin,Resistin, Retinol Binding Globulin RBP, RO SS-A 60 kda, RO/SS-A 52 kda,S100 (human brain) (BB/AB), S100 (human) BB homodimer, Saposin, SCF,SCGF-alpha, SCGF-Beta, SDF-I alpha, SDF-I Beta, Secreted frizzledrelated protein 1 (Sfrp1), Secreted frizzled related protein 2 (Sfrp2),Secreted frizzled related protein 3 (Sfrp3), Secreted frizzled relatedprotein 4 (Sfrp4), Secreted frizzled related protein 5 (Sfrp5),secretin, serum thymic factor, Binding Globulin (SHBG), somatomedin,somatostatin, Somatotropin, s-RankL, substance P, Superoxide Dismutase,TGF alpha, TGF beta, Thioredoxin, Thrombopoietin (TPO), Thrombospondin1, Thrombospondin 2, Thrombospondin 3, Thrombospondin 4, Thrombospondin5, Thrombospondin 6, Thrombospondin 7, thymic humoral factor,thymopoietin, thymosin a1, Thymosin alpha-1, Thymus and activationregulated chemokine (TARC), Thymus-expressed chemokine (TECK),Thyroglobulin Tg, Thyroid Microsomal Antigen, Thyroid Peroxidase,Thyroid Peroxidase TPO, Thyroxine (T4), Thyroxine Binding Globulin TBG,TNFalpha, TNF receptor, Transferin, Transferrin receptor, transforminggrowth factor of the b family, Transthyretin, Triacylglycerol lipase,Triiodothyronine (T3), Tropomyosin alpha, tropomyosin-related kinase(trk), Troponin C, Troponin I, Troponin T, Trypsin, Trypsin Inhibitors,Trypsinogen, TSH, Tweak, Tyrosine Decarboxylase, Ubiquitin, UDPglucuronyl transferase, Urease, Uricase, Urine Protein 1, Urocortin 1,Urocortin 2, Urocortin 3, Urotensin II, Vang-like 1 (Vangl1), Vang-like2 (Vangl2), Vascular Endothelial Growth Factor (VEGF), Vasoactiveintestinal peptide precursor, Vimentin, Vitamine D binding protein, VonWillebrand factor, Wnt1, Wnt10a, Wnt10b, Wnt11, Wnt12, Wnt13, Wnt14,Wnt15, Wnt16, Wnt2, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b,Wnt8a, Wnt8b, Wnt9, Xanthine Oxidase, Clara cell phospholipid bindingprotein, Clostripain, Clusterin, CNTF, Collagen, Collagenase, Collagens(type 1-VI), colony stimulating factor, Complement C1q Complement C3,Complement C3a, Complement C3b-alpha, Complement C3b-beta, ComplementC4, Complement C5, Complement Factor B, Concanavalin A, Corticoliberin,Corticotrophin releasing hormone, C-Reactive Protein (CRP), C-typenatriuretic peptide (Cnp), Cystatin C, D-Dimer, Delta 1, Delta-likekinase 1 (D1k1), Deoxyribonuclease, Deoxyribonuclease I,Deoxyribonuclease II, Deoxyribonucleic Acids, Dersalazine, Dextranase,Diaphorase, DNA Ligase, T4, DNA Polymerase I, DNA Polymerase, T4, EGF,Elastase, Elastase, Elastin, Endocrine-gland-derived vascularendothelial growth factor (EG-VEGF), Elastin Endothelin ElastinEndothelin 1 Elastin Eotaxin Elastin, Epidermal growth factor (EGF),Epithelial Neutrophil Activating Peptide-78 (ENA-78), Erythropoietin(Epo), Estriol, Exodus, Fatty acid-binding proteinFerritin Ferritin,fibroblast growth factor, Fibroblast growth factor 10, Fibroblast growthfactor 11, Fibroblast growth factor 12, Fibroblast growth factor 13,Fibroblast growth factor 14, Fibroblast growth factor 15, Fibroblastgrowth factor 16, Fibroblast growth factor 17, Fibroblast growth factor18, Fibroblast growth factor 19, Fibroblast growth factor 2, Fibroblastgrowth factor 20, Fibroblast growth factor 3, Fibroblast growth factor4, Fibroblast growth factor 5, Fibroblast growth factor 6, Fibroblastgrowth factor 7, Fibroblast growth factor 8, Fibroblast growth factor 9,Fibronectin, focal-adhesion kinase (FAK), Follitropin alfa, GalactoseOxidase, Galactosidase, Beta, gamaIP-10, gastrin, GCP, G-CSF, Glialderived Neurotrophic Factor (GDNF), Glial fibrillary acidic Protein,Glial filament protein (GFP), glial-derived neurotrophic factor familyreceptor (GFR), globulin, Glucose Oxidase, Glucose-6-PhosphateDehydrogenase, Glucosidase, Alpha, Glucosidase, Beta, Glucuronidase,Beta, Glutamate Decarboxylase, Glyceraldehyde-3-Phosphate Dehydrogenase,Glycerol Dehydrogenase, Glycerol Kinase, Glycogen Phosphorylase ISO BB,Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), growthstimulatory protein (GRO), growth hormone, Growth hormone releasinghormone, Hemopexin, hepatic erythropoietic factor (hepatopoietin),Heregulin alpha, Heregulin beta 1, Heregulin beta 2, Heregulin beta 3,Hexokinase, Histone, Human bone morphogenetic protein, Human relaxin H2,Hyaluronidase, Hydroxysteroid Dehydrogenase, Hypoxia-Inducible Factor-1alpha (HIF-1 Alpha), I-309/TCA-3, IFN alpha, IFN beta, IFN gama, IgA,IgE, IgG, IgM, Insulin, Insulin Like Growth Factor I (IGF-I), InsulinLike Growth Factor II (IGF-II), Interferon, Interferon-inducible T cellalpha chemoattractant (I-TAC), Interleukin, Interleukin 12 beta,Interleukin 18 binding protein, Intestinal trefoil factor, IP10, Jagged1, Jagged 2, Kappa light chain, Keratinocyte Growth Factor (KGF), Kiss1,La/SS-B, Lactate Dehydrogenase, Lactate Dehydrogenase, L-, Lactoferrin,Lactoperoxidase, lambda light chain, Laminin alpha 1, Laminin alpha 2,Laminin beta 1 Laminin beta 2, Laminin beta 3, Laminin gamma 1, Laminingamma 2, LD78beta, Leptin, leucine Aminopeptidase, Leutenizing Hormone(LH), LIF, Lipase, liver-cell growth factor, liver-expressed chemokine(LEC), LKM Antigen, TNFbeta, Luciferase, Lutenizing hormone releaseinghormone, Lymphocyte activation gene-1 protein (LAG-1), Lymphotactin,Lysozyme, Macrophage Inflammatory Protein 1 alpha (MIP-1 Alpha),Macrophage-Derived Chemokine (MDC), Malate Dehydrogenase, Maltase,MCP(macrophage/monocyte chemotactic protein)-1, 2 and 3, 4, M-CSF, MEC(CCL28), Membrane-type frizzled-related protein (Mfrp), Midkine, MIF,MIG (monokine induced by interferon gamma), MIP 2 to 5, MIP-1beta, Mp40;P40 T-cell and mast cell growth factor, Myelin Basic ProteinMyeloperoxidase, Myoglobin, Myostatin Growth Differentiation Factor-8(GDF-8), Mysoin, Mysoin LC, Mysoin HC, ATPase, NADase, NAP-2, negativegrowth factor, nerve growth factor (NGF), Neuraminidase, Neuregulin 1,Neuregulin 2, Neuregulin 3, Neuron Specific Enolase, Neuron-SpecificEnolase, neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), Neuturin, NGF,NGF-Beta, Nicastrin, Nitrate Reductase, Nitric Oxide Synthesases,Nortestosterone, Notch 1, Notch 2, Notch 3, Notch 4, NP-1, NT-1 to 4,NT-3 Tpo, NT-4, Nuclease, Oncostatin M, Omithine transcarbamoylase,Osteoprotegerin, Ovalbumin, Oxalate Decarboxylase, P16, Papain, PBP,PBSF, PDGF, PDGF-AA, PDGF-AB, PDGF-BB, PEDF, Pepsin, Peptide YY (PYY),Peroxidase, Persephin, PF-4, P-Glycoprotein, Phosphatase, Acid,Phosphatase, Alkaline, Phosphodiesterase I, Phosphodiesterase II,Phosphoenolpyruvate Carboxylase, Phosphoglucomutase, Phospholipase,Phospholipase A2, Phospholipase A2, Phospholipase C, PhosphotyrosineKinase, Pituitary adenylate cyclase activating polypeptide, PlacentalLactogen, Plakoglobin, Plakophilin, Plasma Amine Oxidase, Plasma retinolbinding protein, Plasminogen, Pleiotrophin (PTN), PLGF-1, PLGF-2,Pokeweed Antiviral Toxin, Prealbumin, Pregnancy assoc Plasma Protein A,Pregnancy specific beta 1 glycoprotein (SPI), Prodynorphin,Proenkephalin, Progesterone Proinsulin, Prolactin,Pro-melanin-concentrating hormone (Pmch), Pro-opiomelanocortin,proorphanin, Prostate Specific Antigen PSA, Prostatic Acid PhosphatasePAP, Prothrombin, PSA-A1, Pulmonary surfactant protein A, PyruvateKinase, Ranpimase, RANTES, to Reelin, Renin, Resistin, Retinol BindingGlobulin RBP, RO SS-A 60 kda, RO/SS-A 52 kda, S100 (human brain)(BB/AB), S100 (human) BB homodimer, Saposin, SCF, SCGF-alpha, SCGF-Beta,SDF-1 alpha, SDF-1 Beta, Secreted frizzled related protein 1 (Sfrp 1),Secreted frizzled related protein 2 (Sfrp2), Secreted frizzled relatedprotein 3 (Sfrp3), Secreted frizzled related protein 4 (Sfrp4), Secretedfrizzled related protein 5 (Sfrp5), secretin, serum thymic factor,Binding Globulin (SHBG), somatomedin, somatostatin, Somatotropin,s-RankL, substance P, Superoxide Dismutase, TGF alpha, TGF beta,Thioredoxin, Thrombopoietin (TPO), Thrombospondin 1, Thrombospondin 2,Thrombospondin 3, Thrombospondin 4, Thrombospondin 5, Thrombospondin 6,Thrombospondin 7, thymic humoral factor, thymopoietin, thymosin a1,Thymosin alpha-1, Thymus and activation regulated chemokine (TARC),Thymus-expressed chemokine (TECK), Thyroglobulin Tg, Thyroid MicrosomalAntigen, Thyroid Peroxidase, Thyroid Peroxidase TPO, Thyroxine (T4),Thyroxine Binding Globulin TBG, TNFalpha, TNF receptor, Transferin,Transferrin receptor, transforming growth factor of the b family,Transthyretin, Triacylglycerol lipase, Triiodothyronine (T3),Tropomyosin alpha, tropomyosin-related kinase (trk), Troponin C,Troponin I, Troponin T, Trypsin, Trypsin Inhibitors, Trypsinogen, TSH,Tweak, Tyrosine Decarboxylase, Ubiquitin, UDP glucuronyl transferase,Urease, Uricase, Urine Protein 1, Urocortin 1, Urocortin 2, Urocortin 3,Urotensin II, Vang-like 1 (Vangl1), Vang-like 2 (Vangl2), VascularEndothelial Growth Factor (VEGF), Vasoactive intestinal peptideprecursor, Vimentin, Vitamine D binding protein, Von Willebrand factor,Wnt1, Wnt10a, Wnt10b, Wnt11, Wnt12, Wnt13, Wnt14, Wnt15, Wnt16, Wnt2,Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9and Xanthine Oxidase.

Following the genetic modification process, the tissue sample may bethen analyzed in order to verify the expression of the gene of interestby the tissue sample. This could be done by any method known in the art,for example by ELISA detection of proteins or Northern blot for RNA. Theefficacy of a particular expression vector system and method ofintroducing nucleic acid into a cell can be assessed by standardapproaches routinely used in the art. For example, DNA introduced into acell can be detected by a filter hybridization technique (e.g., Southernblotting) and RNA produced by transcription of introduced DNA can bedetected, for example, by Northern blotting, RNase protection or reversetranscriptase-polymerase chain reaction (RT-PCR). The gene product canbe detected by an appropriate assay, for example by immunologicaldetection of a produced protein, such as with a specific antibody, or bya functional assay to detect a functional activity of the gene product,such as an enzymatic assay. If the gene product of interest to beexpressed by a cell is not readily assayable, an expression system canfirst be optimized using a reporter gene linked to the regulatoryelements and vector to be used. The reporter gene encodes a gene productwhich is easily detectable and, thus, can be used to evaluate efficacyof the system. Standard reporter genes used in the art include genesencoding .beta.-galactosidase, chloramphenicol acetylm transferase,luciferase, GFP/EGFP and human growth hormone.

The invention contemplates, in one aspect, the use of the geneticallymodified DTMO for transplantation in an organism. As used herein theterms “administering”, “introducing”, “implanting” and “transplanting”may be used interchangeably and refer to the placement of the DTMO ofthe invention into a subject, e.g., an autologous, allogeneic orxenogeneic subject, by a method or route which results in localizationof the DTMO at a desired site. The DTMO is implanted at a desiredlocation in the subject in such a way that at least a portion of thecells of the DTMO remain viable. In one embodiment of this invention, atleast about 5%, in another embodiment of this invention, at least about10%, in another embodiment of this invention, at least about 20%, inanother embodiment of this invention, at least about 30%, in anotherembodiment of this invention, at least about 40%, and in anotherembodiment of this invention, at least about 50% or more of the cellsremain viable after administration to a subject. The period of viabilityof the cells after administration to a subject can be as short as a fewhours, e.g., twenty-four hours, to a few days, to as long as a few weeksto months or years. To facilitate transplantation of the cellpopulations within a tissue which may be subject to immunological attackby the host, e.g., where xenogenic grafting is used, such as swine-humantransplantations, the DTMO may be inserted into or encapsulated bybiocompatible immuno-protected material such as rechargeable,non-biodegradable or biodegradable devices and then transplanted intothe recipient subject. Gene products produced by such cells/tissue canthen be delivered via, for example, polymeric devices designed forcontrolled delivery of compounds, e.g., drugs, including proteinaceousbiopharmaceuticals. A variety of biocompatible polymers (includinghydrogels, for example), including both biodegradable and non-degradablepolymers, can be used to form an implant for the sustained release of agene product of the cell populations of the invention at a particulartarget site. The generation of such implants is generally known in theart. See, for example, Concise Encyclopedia of Medical & DentalMaterials, ed. By David Williams (MIT Press: Cambridge, Mass., 1990);the Sabel et al. U.S. Pat. No. 4,883,666; Aebischer et al. U.S. Pat. No.4,892,538; Aebischer et al. U.S. Pat. No. 5,106,627; Lim U.S. Pat. No.4,391,909; and Sefton U.S. Pat. No. 4,353,888. Cell populations withinthe DTMO of the present invention can be administered in apharmaceutically acceptable carrier or diluent, such as sterile salineand aqueous buffer solutions. The use of such carriers and diluents iswell known in the art.

The secreted protein such as, for example, without limitation, may beany protein according to the embodiments of the invention describedabove. The protein of interest may be, in one embodiment of thisinvention, erythropoietin. In another embodiment of this invention, themethod of the invention may be used for the expression and secretion ofeach and any protein known in the art and combinations thereof. Inaddition, the method of the invention may be used for the expression ofRNA molecules (sense or antisense)

Alternatively, the DMO, which includes genetically modified cells can bekept in vitro and the therapeutic agent, left in the supernatant mediumsurrounding the tissue sample, can be isolated and injected or appliedto the same or a different subject.

Alternatively or additionally, a dermal micro-organ which includes agenetically modified cell can be cryogenically preserved by methodsknown in the art, for example, without limitation, gradual freezing(0.degree. C., −20.degree. C., −80.degree. C., −196.degree. C.) in DMEMcontaining 10% DMSO, immediately after being formed from the tissuesample or after genetic alteration.

In accordance with an aspect of some embodiments of the invention, themounts of tissue sample including a genetically modified cell(s) to beimplanted are etermined from one or more of: Corresponding amounts ofthe therapeutic agent of interest outinely administered to such subjectsbased on regulatory guidelines, specific clinical protocols orpopulation statistics for similar subjects. Corresponding amounts of thetherapeutic agent such as protein of interest specifically to that samesubject in the case that he/she has received it via injections or otherroutes previously. Subject data such as weight, age, physical condition,clinical status. Pharmacokinetic data from previous tissue sample whichincludes a genetically modified cell administration to other similarsubjects. Response to previous tissue sample which includes agenetically modified cell administration to that subject.

In accordance with an aspect of some embodiments of the invention, onlysome of the DTMOs are used in a given treatment session. The remainingDTMOs may be returned to maintenance (or stored cryogenically orotherwise), for later use.

There is thus provided in accordance with an embodiment of theinvention, a method of determining the amount of a therapeutic dermalmicro-organ to be implanted in a patient, the method includingdetermining a secretion level of a therapeutic agent by a quantity ofthe DTMO in vitro; estimating a relationship between in vitro productionand secretions levels and in vivo serum levels of to the therapeuticagent; and determining an amount of DTMO to be implanted, based on thedetermined secretion level and the estimated relationship. Optionally,the relationship is estimated based one or more factors chosen from thefollowing group of factors:

a) Subject data such as weight, age, physical condition, clinicalstatus;

b) Pharmacokinetic data from previous DTMO administration to othersimilar subjects; and

c) Pharmacokinetic data from previous DTMO administration to thatsubject.

Optionally, the relationship is estimated based on at least two of saidfactors. Optionally, the relationship is based on three of said factors.

In an embodiment of the invention, determining an amount of a DTMO to beimplanted in a patient is also based on one or both of:

corresponding amounts of the same therapeutic protein routinelyadministered to such subjects based on regulatory guidelines, specificclinical protocols or population statistics for similar subjects; and

corresponding amounts of the same therapeutic agent specific to thatsame subject in the case the subject has received it previously viainjections or other administration routes.

In an embodiment of the invention, the method includes preparing anamount of DTMO for implantation, in accordance with the determinedamount.

There is also provided in accordance with an embodiment of theinvention, method of adjusting the dosage of a therapeutic agentproduced by a DTMO implanted in a subject and excreting a therapeuticagent, including (a) monitoring level of therapeutic agent in thesubject; (b) comparing the level of agent to a desired level; (c) if thelevel is lower than a minimum level, then implanting additional DTMO;(d) and if the level is higher than a maximum level, then inactivatingor removing a portion of the implanted DTMO. Optionally, the methodincludes periodically repeating (a)-(d). Alternatively or additionally,inactivating or removing consists of removing a portion of the implantedDTMO. Optionally, removing includes surgical removal. Alternatively oradditionally, inactivating or removing includes inactivating.Optionally, inactivating includes killing a portion of the implantedDTMO. Optionally, inactivating includes ablating a portion of theimplanted DTMO.

As described above with reference to FIG. 1, at least part of theprocess of sustaining the DMO during the genetic alteration, as well asthe genetic alteration itself, may be performed in a bioreactor, asdescribed below.

According to some embodiments of the invention, the bio-reactor may havesome or all of the following properties:

a) Allow for the provision of nutrients and gasses to the surfaces ofthe DMO so that they may diffuse into the DMO and the DMO may remainviable. Thus, significant areas and volumes of the DMO may not beblocked from coming into contact with a surrounding fluid.

b) Allow for the maintenance of the DMO at a desired temperature.

c) Allow for the maintenance of a desired pH and gas composition in thevicinity of the DMO.

d) Allow for the removal of waste products from the DMO and/or from thebio-reactor.

e) Allow for a simple method of inserting the genetically modifyingvector without substantial danger that the inserting vector willcontaminate the surroundings.

f) Allow for the removal of excess unused vector.

g) Allow for measurement of the amount of therapeutic agent generated.

h) Allow for removal of substantially sterile therapeutic agent.

i) Allow for easy insertion of the DMO and removal of all or measuredamounts of DTMO.

Reference in now made to FIG. 22, which schematically illustrates asystem 2207 for processing a harvested DMO 2204, according to someexemplary embodiments of the invention.

According to some exemplary embodiments of the invention, system 2207may include a bioreactor 2200 having one or more processing chambers2202, each adapted to accommodate a DMO 2204. Bioreactor 2200, which inone exemplary embodiment has a number of chambers equal to the number ofDMOs harvested from a particular subject, may be adapted to provide oneor more of processing chambers 2202 with a suitable fluid or fluids,e.g., a growth medium, from a local fluid reservoir 2208 and/ordischarge the fluid of one or more of processing chambers 2202, e.g., toa waste container 2210, as described below. The fluid may be supplied toreservoir 2208 via an inlet line 2242, e.g., connected by a sterileconnector 2258 to reservoir 2208, as described below.

DMO 2204 may be transferred to chamber 2202 using a cutting tool usedfor harvesting DMO 2204, e.g., as described above. The DMO transfer intochamber 2202 may be preferably performed directly after harvesting DMO2204 and while maintaining sterile conditions. Processing chamber 2202may include a DMO insertion port 2201 adapted for receiving DMO 2204.For example, port 2201 may include a sterile septum interface capable ofreceiving a blunt cannula, e.g., a SafeLine™ Injection Site marketed byB. Braun Medical Inc. Once the tip of the cutting tool is insertedthrough the septum, DMO 2204 may be gently flushed into chamber 2202 ina generally sterile manner, e.g., using a syringe connected to the backend of the cutting tool. According to one exemplary embodiment, DMO 2204may be flushed into a medium bath 2206 within chamber 2202.Alternatively, if, for example, DMO 2204 was harvested with an innerguide, e.g., described above, a lid 2232 fitted over chamber 2202, e.g.,as described below, may be removed, DMO 2204 may be gently removed fromthe inner guide and placed within chamber 2202, and lid 2232 may bereturned and sealed over chamber 2202 to maintain sterility of chamber2202.

Bioreactor 2200 may be adapted to apply, e.g., in a generally identicalmanner, one or more processes to DMOs being accommodated within at leastsome of the processing chambers. According to exemplary embodiments ofthe invention, bioreactor 2200 may be adapted to fluidically separatethe contents of one or more of the processing chambers from the contentsof one or more other processing chambers, as described below.

According to exemplary embodiments of the invention, bioreactor 2200 mayalso include a mechanism for controlling the flow of a fluid into and/orout of processing chamber 2202, as described below.

According to an exemplary embodiment, bioreactor 2200 may include asterile buffer 2222 fluidically connected to a non-sterile syringe pump2214, which may be adapted to inject air into buffer 2222 and/ordischarge air from buffer 2222 in a sterile manner, e.g., via a sterilefilter 2220, e.g. a 0.45 .mu.m pore air filter. Bioreactor 2200 may alsoinclude a control valve 2212 able to be moved between at least fourpositions, e.g., an inlet-buffer position wherein inlet reservoir 2208is fluidically connected to buffer 2222, an outlet-buffer positionwherein waste container 2210 is fluidically connected to buffer 2222, achamber-buffer position wherein chamber 2202 is fluidically connected tobuffer 2222, and/or a no-connection position wherein buffer 2222,chamber 2202, inlet reservoir 2208, and waste container 2210 arefluidically disconnected from each other. A piston 2226 may connectbetween valve 2212 and a motor 2224 adapted to move valve 2212 betweenthe different positions. Optionally, a bellows diaphragm 2228 may befitted over piston 2226 such that there is substantially no transfer ofnon-sterile air from into the sterile buffer 2222, e.g., during motionof piston 2226.

System 2201 may also include a motor 2216 to actuate a plunger 2218 ofsyringe pump 2214. If bioreactor 2200 includes more than one chamber,then either one motor may be implemented for simultaneously actuatingeach one of the plungers associated with the chambers, or a plurality ofmotors may be implemented, each able to actuate one or more of theplungers.

According to exemplary embodiments of the invention, system 2201 mayinclude a controller 2286 able to control the operation of motor 2216and/or motor 2224, e.g., as described below.

According to exemplary embodiments of the invention, fluid fromreservoir 2208 may be controllably transferred into chamber 2202, e.g.,in order to fill chamber 2202. For example, controller 2286 may activatemotor 2224 to position valve 2212 at the inlet-buffer position, andcontrollably activate motor 2216 such that syringe pump 2214 evacuates apredetermined quantity of air from buffer 2222. As a result apredetermined volume of fluid corresponding to the predetermined volumeof air may be “drawn” from inlet reservoir 2208 into buffer 2222.Controller 2286 may then controllably activate motor 2224 to move valve2212 to the chamber-buffer position, and controllably activate motor2216 such that syringe pump 2214 discharges the fluid of buffer 2222into chamber 2202. In a similar manner, the syringe pump and controlvalve may be controlled to discharge the contents of chamber 2202, or apartial amount thereof, into waste container 2210.

According to some exemplary embodiments of the invention, the fluid inchamber 2202 may be controllably stirred and/or mixed, e.g., in order toassist viral transduction and/or any other ex-vivo maintenance procedureapplied to DMO 2204. For example, controller 2286 may controllablyactivate motor 2216 and/or motor 2224, e.g., as described above, toperiodically discharge the fluid, or a part thereof, from chamber 2202into buffer, and thereafter to inject the fluid in buffer 2222 back intochamber 2202.

According to some exemplary embodiments of the invention, air may beused to purge fluid located in one or more “passage lines”, e.g.,fluidically connecting between inlet reservoir 2208, waste container2210 and/or chamber 2202, for example, in order to “flush” the passagelines after transferring fluid to/from chamber 2202, inlet reservoir2208, and/or buffer 2222. This aspect may be useful, for example, inorder to reduce a “dead volume” of fluid, which may be “trapped” in oneor more of the passage lines. For example, controller 2286 maycontrollably activate motor 2216 to move syringe plunger 2218 such thata predetermined volume of air is drawn into buffer 2222, before drawingthe fluid from reservoir 2208 into buffer 2222. Buffer 2222 may have ageometry such that the air will rise above the fluid within buffer 2222,such that upon actuation of syringe pump 2214 the fluid in buffer 2222may be discharged first, followed by the air, which will act to flushthe passage lines of some or all of the fluid remaining therein.

According to some exemplary embodiments of the invention, a bottomsurface 2230 of chamber 2202 may include a plurality of holes, or amesh-like pattern, e.g., configured to enable the fluid to betransferred into and/or out of chamber 2202 in a substantially uniformmanner, and/or to allow discharging substantially most of the fluid fromchamber 2202. This configuration may also enable reducing the occurrenceof “dead-spots”, i.e., areas of chamber 2202 in which the fluid remainsstagnant and/or is not refreshed.

According to some exemplary embodiments, lid 2232 may be a removablesterile lid, such as a membrane affixed by a detachable adhesive,silicon plug material, or the like. Lid 2232 may be adapted to maintaina sterile “barrier” between chamber 2202 and the environment.Optionally, a sterile air filter 2234, e.g., a 0.451 .mu.m pore airfilter, may be implemented to fluidically connect chamber 2202 and theenvironment, thus enabling equilibration of pressures while maintaininga sterile bather between chamber 2202 and the environment.Alternatively, lid 2232 may include a “breathable” material, such thatpressure equilibration may be enabled through lid 2232.

Reservoir 2208 and/or waste container 2210 may be commonly connected,e.g., via one or more manifolds (not shown), to one or more ofprocessing chambers 2202 for a specific subject. Alternatively, inletreservoir 2208 and/or waste container 2210 may be individually connectedto each one of the processing chambers. Inlet reservoir 2208 and/orwaste container 2210 may include a mechanism for equilibrating pressurein a sterile manner. For example, inlet reservoir 2208 and/or wastecontainer 2210 may be fluidically connected to the environment via asterile air filter 2236 and/or a sterile air filter 2238, respectively.Filter 2236 and/or filter 2238 may include, for example, a 0.45 .mu.mpore air filter. Alternatively, waste container 2210 may include anexpandable waste container, such that no pressure equilibration isrequired and, therefore, no sterile air filter need be used for it.

According to an exemplary embodiment of the invention, bioreactor 2200may be adapted to enable direct injection of fluid or discharging offluid to/from chamber 2202. A sampling septum port 2240 may be used, forexample, for direct injection of viral vector fluid, or for sampling ofgrowth medium to test for various bioreactor parameters, such as ELISA,glucose uptake, lactate production or any other indicative parameter.Septum port 2240 may include a standard silicon port adapted for needleinsertion or a cannula port, e.g., as described above with reference toDMO insertion port 2201. A syringe (not shown) may be detachablyinserted through septum port 2240. The syringe may be driven by a motor,e.g., similar to motor 2216, which may be activated manually orautomatically, e.g., by controller 2286.

According to exemplary embodiments of the invention, at least some, andin some exemplary embodiments all, components of bioreactor 2200 may bemaintained at predetermined conditions, e.g., incubator conditions,including a temperature of approximately 37.degree. C., a gaseousatmosphere of approximately 90-95% air and approximately 5-10% CO.sub.2,and/or a relatively high degree of humidity, e.g., 85-100%. According toone exemplary embodiment, only chamber 2202 may be maintained in theincubator conditions. As described above, these incubator conditions maybe required, e.g., for maintaining the vitality of the DMO tissueculture.

According to exemplary embodiments of the invention, a fluid supplyarrangement may be implemented for supplying fluid to inlet line 2242from at least one fluid tank, e.g., fluid tanks 2244 and 2246. In oneexemplary embodiment, tanks 2244 and 2246 may contain the same fluid,e.g., a growth medium, in which case one tank may be used as a backupreservoir for the other tank. In another exemplary embodiment, tanks2244 and 2246 may contain two different types of fluids, such as twotypes of growth medium to be used at different stages of DMO processing.Tank 2244 and/or tank 2246 may include a sterile air filter toequilibrate pressure in a sterile manner, e.g., as described above withreference to reservoir 2208. Alternatively, tank 2244 and/or tank 2246may include a collapsible tank, e.g., a sterile plastic bag as is knownin the art.

According to exemplary embodiments of the invention, each of tanks 2244and 2246 may be fluidically connected to a combining connector 2254 viaa valve 2252, e.g., a pinch valves, a septum port connector 2248 and apenetration spike 2250. Connector 2254 may include, for example aY-shaped or a T-shaped connector as is known in the art. Valve 2252 maybe adapted to control the flow of fluid from tank 2244 and/or tank 2246to connector 2254. A pump, e.g., a peristaltic pump, 2256 may be locatedbetween connector 2254 and connector 2258, along inlet line 2242.Controller 2286 may be used to control the amount and/or flow-rate ofthe fluid provided to reservoir 2208 by controllably actuating motor2257 and/or valves 2252.

According to one exemplary embodiment, the fluid contained within tanks2244 and/or tank 2246 may have a storage shelf life of 9 days atrefrigerated 4.degree. C. conditions. Thus, a refrigeration system (notshown) may be employed to maintain the fluid of tanks 2244 and/or 2246at a temperature, which may be lower than the incubation temperature ofreservoir 2208. Accordingly, inlet line 2242 may pass through aninterface between refrigerator conditions to incubator conditions. Afterthe shelf life has expired, tank 2244 and/or tank 2246 may be replacedby new tanks.

According to an exemplary embodiment, at least some of the elements ofbioreactor 2200 may be formed of disposable sterile plastic components.According to these embodiments, bioreactor apparatus 2200 may include asingle-use sterilely packaged bioreactor apparatus, which may beconveyed to a DMO harvesting site and may be opened in a sterileenvironment and prepared on site such that growth medium is injectedinto each bioreactor chamber 2202. The tool used for harvesting the DMOsmay be inserted through the DMO insertion ports 2201 to flush the DMOsinto chambers 2202 in a sterile fashion, as described above. Bioreactorapparatus 2200 may be transported, e.g., under incubator conditions, toa processing site where it may be connected to other components ofsystem 2207, e.g., connector 2258, motors 2216 and/or 2224, pinch valves2252, and/or peristaltic pump 2256. Controller 2286 may then control themaintenance and transduction of the DMOs during the entire ex-vivoprocessing in which the DTMO is produced from the harvested DMO. Thedosage needed for the specific subject may be determined by use of thepharmacokinetic model, e.g., as described herein. Bioreactor apparatus2200 may then be detached from system 2207 and transported, e.g., underincubator conditions, to the site of implantation. In order to implant aspecific DTMO, e.g., according to the implantation methods describedabove, bioreactor chamber 2202 for the specific DTMO may be opened byremoving lid 2232 and the DTMO may be removed from the chamber.

EXAMPLES Example 1

In Vitro Secretion Levels of Human Erythropoietin by DTMO-hEPO

Experiments were conducted to assay the variability of in vitro hEPOsecretion level between DTMOs-hEPO obtained from different human skinsamples.

Experimental Procedure

DTMO-hEPO was prepared (in triplicates) from skin samples obtained fromsix different human subjects and hEPO secretion levels were measured atvarious point in time, as indicated in FIG. 4, after the viral vectorwas washed.

Experimental Results

The DTMO-hEPO secretion levels were similar among the different humanskin samples. In addition, the DTMO-HEPO secretion levels were similarto the secretion levels of hEPO previously obtained from split thicknessTMO-hEPO (data not shown).

Example 2 Histology

In order to verify that the DTMO contains mainly dermal components, ahistological analysis was performed. MOs were prepared from either splitthickness skin or dermal skin samples and histological analysis wasperformed by a dermato-pathologist. As can be seen on the left side ofFIG. 5, the DTMO contains dermal layers and dermal components withoutresidual basal and/or epidermal layers. In comparison, the splitthickness TMO, shown on right side of FIG. 5, contains all the skinlayers including the basal and epidermal layers.

Example 3 Immunocytochemistry Studies

To study which cells are transduced in the DTMO-hEPO tissue, ahistological immunohistochemistry analysis of DTMO-hEPO was performed onday 9 post-harvesting, using an anti-hEPO monoclonal antibody (1:20dilution). Analysis revealed strong staining of dermal to fibroblasts,as shown in FIG. 6. The staining was spread throughout the entire DTMO.

Example 4 Comparison of Long Term hEPO Hematopoietic Activity in SCIDMice Derived from DTMO-hEPO and Entire TMO

An experiment was performed to examine and compare the long term effectsof subcutaneously implanted DTMO-hEPO and Split thickness derivedTMO-HEPO in SCID mice.

Experimental Procedure

Human DTMO-hEPO and human Split thickness derived TMO-hEPO were preparedand implanted subcutaneously in two groups of SCID mice (five mice pergroup). A control group was implanted with human DTMO and Splitthickness derived TMO transduced with an Ad/lacZ viral vector.

Experimental Results

As is shown in FIG. 7, similar secretion levels and physiologicalresponse were identified in the two experimental groups while, asexpected, the control group mice had no hEPO in their blood.

In all experimental groups, an elevation of hematocrit can be seen asearly as 15 days post-implantation and is maintained for more than 5months, while the MO/lacZ control mice do not show such an elevation inhematocrit level. DTMO-HEPO seems to result in similar secretion levelsfor similar time periods when compared to split thickness derivedTMO-hEPO.

Example 5 DTMO-hEPO Do not Form Keratin Cysts when ImplantedSub-Cutaneously Experimental Procedure

DTMO-hEPO and split thickness derived TMO-hEPO were implanted S.C. inSCID mice and keratin cyst formation was monitored by clinical andhistological analysis.

Experimental Results

As can be clearly seen in FIG. 8, keratin cyst formation was observedwhile implanting the to split thickness derived TMO-hEPO 76 and 141 dayspost implantation. In contrast, no cyst formation was observed in SCIDmice with the DTMO-hEPO 113 days post implantation.

Example 6 Split Thickness Derived and DMO Integration in Healthy HumanSubjects Experimental Procedure

Human Dermal MO and human split thickness derived Split thicknessderived TMO were obtained using a commercially available dermatome(Aesculap GA 630). Prior to harvesting, topical and local anesthesia forboth the donor and recipient site were performed using Emla lotion(topical anesthesia) and subcutaneous injections of Marcain+Adrenalin(local anesthesia).

Two types of skin samples were harvested in order to produce humanDermal MO and human split thickness derived MO. For human splitthickness derived MO, a strip of healthy skin was excised from the lowerpart of the abdomen. From this skin section, six linear MOs wereprepared as previously described. Simultaneously, slits of specificdimensions were made in the implantation site using an adjustable slitmaker, and MOs were grafted shortly after into the skin slits. Forpreparing Human Dermal MO, skin was harvested in two steps. First, askin flap of 200 .mu.m in depth was harvested and kept on moist gauze.From this harvest site, a 1 mm deep dermis skin strip was harvested.Following skin harvesting, the 200 .mu.m skin flap was placed back onthe donor site serving as a biological dressing. From the dermis stripharvested above, four dermal MOs were prepared utilizing an identicalprocedure as for the split thickness derived Split thickness derived TMOMO. The human Dermal MO were implanted subcutaneously shortly after,using a trocar. The donor and implantation sites were dressed usingBioclusive™ transparent membrane (Johnson& Johnson, USA). After one weekthe dressing was changed and the implants were examined to check graftintegration. Two to three weeks following the MO implantation, thescheduled abdominoplasty procedure was performed and a section of skin,including the graft and implantation area was excised. A clinicalevaluation was performed on the graft area including photographs andhistological examination to determine MO integration.

Experimental Results

A clinical inspection, which was performed one week after implantation,and histological analysis, which was performed soon after abdominoplasty(2-3 weeks after grafting), revealed excellent integration of thegrafted MOs into the skin slits and at the dermal MOs subcutaneousimplantation sites (FIG. 9). No indication of inflammation or swellingwas found on either split thickness derived MOs that were implanted intothe slits or Dermal Mos that were implanted subcutaneously.

Example 7 Autologous Implantation of Miniature Swine Skin Linear SplitThickness TMOs, Expressing Human Erythropoietin (hEPO into ImmunoCompetent Animals)

Linear (30.6 mm long and 0.6 micrometer wide) miniature swine (Sinclarswine) skin micro-organs were prepared from fresh skin tissue samplesobtained from live animals under general anesthesia procedures. Tissuesamples of 0.9-1.1 mm split skin thickness (depth) were removed using acommercial dermatome (Aesculap GA630) and cleaned using DMEM containingglutamine and Pen-Strep in Petri dishes (90 mm).

In order to generate the linear micro-organs, the above tissue sampleswere cut by a press device using a blade structure as described above,into the desired dimensions: 30.6 mm times.600 micrometers. Theresulting linear micro-ograns were placed, one per well, in a 24-wellmicro-plate containing 500 .mu.l per well of DMEM (BiologicalIndustries—Beit Haemek) in the absence of serum under 5% CO.sub.2 at37.degree. C. for 24 hours. Each well underwent a transduction procedurein order to generate a miniature swine skin therapeutic micro-organ (pigskin-TMO) using an adeno viral vector (1.times.10.sup.10 IP/ml) carryingthe gene for human erythropoietin (Adeno-hEPO) for 24 hours while theplate was agitated. The medium was changed every 2-4 days and analyzedfor the presence of secreted HEPO using a specific ELISA kit (Cat. #DEP00, Quantikine IVD, R&D Systems).

The above described miniature swine skin hEPO linear TMOs were implantedboth sub-cutaneously and grafted as skin grafts in several immunecompetent miniature swines (in two of the miniature swine, the TMOs-hEPOwere implanted subcutaneously, and in two different miniature swine,TMOs-hEPO were grafted in 1 mm deep slits). A sufficient number ofTMOs-hEPO were implanted in each miniature swine so that their combinedpre-implantation secretion level in each pig was approximately 7micrograms per day. Elevated serum HEPO levels (FIG. 3A) determined byan ELISA assay and reticulocyte count elevation were obtained for sevendays after implantation.

Example 8 Secretion Levels from Human Dermal MO Transduced by DifferentViral Vectors Experimental Procedures

Human DMO were produced from abdominal skin samples obtained from theskin of healthy donors. Transduction was done using research grade viralvectors encoding recombinant protein diluted to working concentrations.To remove the viral vector that had not entered the cells, washes usingculture media were performed. The maintenance steps followed a standardprocedure using culture medium for the duration of the experiment. Otherparameters such as well plates, media volume, and incubation conditionsremained unchanged throughout the experiment.

Culture media used was DMEM-HEPES, Gentamycin 50 μg/ml, Fungizone 2.5μg/ml, and L-Glutamine 2 mM, further supplemented with 10% FBS or 10%SSS. Cultures were maintained In 10% CO2, at 32° C. Media was changedand collected for analyses by ELISA every three to four days, and levelsof the secreted recombinant proteins were measured.

TDMO-Expressing Erythropoietin; Adeno-Associated Virus Serotype 1

Human Dermal core MOs were prepared in a sterile hood using a14G needle.The DMO's were washed three times with culture media in a 10 cm plateand divided into individual wells with 1 ml media, in each of three 24well/plate. A 16-24 hour recovery period in culture followed platinginto individual wells. Before transduction the vector stocks werediluted in culture medium and the DMO's were transduced in 48well-plates with 100 μl diluted viral vector AAV1-EPO (adeno associatedvirus serotype 1—Erythropoietin). The DNA sequence for EPO is either thewild type EPO sequence or the Optimized EPO sequence. Both encode theexact same amino acid sequence as the wild type gene sequence howeverthe DNA sequence of the optimized gene was altered and utilizesoptimized codon sequences that could be better utilized by the mammaliancells.

TDMO-Expressing Interferon α; Helper-Dependent Adenovirus

Dermal core MOs were prepared in a sterile hood using a14G needle andintra-dermal 22G guiding needle. The DMO's were washed three times withculture media in a 10 cm plate and divided into individual wells with 1ml media, in each of three 24 well/plate. A 16-24 hour recovery periodin culture followed plating into individual wells. Before transductionthe vector stocks were diluted in culture media and the DMO's weretransduced in 48 well-plates with 100 μl diluted viral vectorHDAd-CAG-IFNα (HDAd-Helper Dependent Adenovirus; CAG-CMV earlyenhancer/chicken β actin promoter; IFNα-Interferon alpha). The DNAsequence for IFN alpha is an optimized sequence that encodes the exactsame amino acid sequence as the wild type gene but utilizes codonoptimized sequences.

TDMO-Expressing α-1-Antitrypsin; Helper-Dependent Adenovirus

Dermal core MOs were prepared in a sterile hood using a14G needle andintra-dermal 22G guiding needle. The DMO's were washed three times withDMEM-HEPES+10% SSS (Serum Substitute Supplement) in 10 cm plate anddivided into individual wells with 1 ml media supplemented with 10% SSS,in a 24 wellplate. A 16-24 hour recovery period in culture followedplating into individual wells. Before transduction the vector stockswere diluted in culture media and the DMO's were transduced in 48well-plates with 100 μl diluted viral vector HDAd-PGK-AAT (HelperDependent Adenovirus; PGK-phosphoglycerate kinase promoter;AAT-alpha-1-antitrypsin).

TDMO-Expressing Erythropoietin; Helper-Dependent Adenovirus

Dermal core MOs were prepared in a sterile hood using a14G needle. TheDMO's were washed three times with culture media in a 10 cm plate anddivided into individual wells with 1 ml media, in each of three 24well/plate. A 16-24 hour recovery period in culture followed platinginto individual wells. Before transduction the vector stocks werediluted in culture media the DMO's were transduced in 48 well-plateswith 100 μl diluted viral vector HD-S/MAR-CAG-EPO (HDAd-Helper DependentAdenovirus; S/MAR-Scaffold/Matrix attachment region; CAG-CMV earlyenhancer/chicken β actin promoter; EPO-erythropoietin).

Experimental Results

As shown in FIGS. 23, 24, 25 and 26, TDMO were produced followingtransduction of DMO by different viral vectors. FIG. 23 demonstratessignificant protein secretion of hEPO expressed from either a wild-typeEPO gene sequence or an optimized EPO sequence, over an extended timecourse with continued secretion maintained through day 154. FIG. 24demonstrates significant protein secretion of α-interferon over anextended time course, with continuous secretion maintained through day295. FIG. 25 demonstrates high levels of secretion of α-1-antitrypsinusing a gutless adeno virus. FIG. 26 demonstrates dependence of TDMOsecretion levels of a protein product on viral components, note theincreased levels of erythropoietin secretion in the presence ofcis-acting S/MAR elements.

It will thus be clear, the present invention has been described usingnon-limiting detailed descriptions of embodiments thereof that areprovided by way of example and that are not intended to limit the scopeof the invention. For example, only a limited number of genetic changeshave been shown. However, based on the methodology described herein inwhich live tissue is replanted in the body of the patient, and theviability of that tissue in the body after implantation, it is clearthat virtually any genetic change in the tissue, induced by virtuallyany known method will result in secretions of target proteins or othertherapeutic agents in the patient.

Variations of embodiments of the invention, including combinations offeatures from the various embodiments will occur to persons of the art.The scope of the invention is thus limited only by the scope of theclaims. Furthermore, to avoid any question regarding the scope of theclaims, where the terms “comprise” “include,” or “have” and theirconjugates, are used in the claims, they mean “including but notnecessarily limited to”.

1. A method of inducing a local or systemic physiological effect in a subject comprising implanting in the subject a genetically modified dermal micro-organ expressing at least a recombinant factor VIII protein or an equivalent thereof, wherein said dermal micro-organ is an explant of living tissue consisting essentially of a plurality of dermal components and lacking a complete epidermal layer and maintaining the micro-architecture and three-dimensional structure of the dermal tissue from which they are obtained, having dimensions selected so as to enable passive diffusion of adequate nutrients and gases to cells of said dermal micro-organ and diffusion of cellular waste out of said cells so as to minimize cellular toxicity and concomitant death due to insufficient nutrition and accumulation of waste in said dermal micro-organ, wherein at least some of said cells of said dermal micro-organ express and secrete at least a portion of said recombinant factor VIII protein or equivalent thereof, and wherein said implanting induces a local or systemic physiological effect in a subject.
 2. The method of claim 1, wherein said genetically modified dermal micro-organ further comprises an in vivo demarcation.
 3. The method of claim 2, wherein said in vivo demarcation comprises an ink or stain on the peripheral surface of said micro-organ, or a green fluorescent protein (GFP) gene or a luciferase reporter gene expressed by said micro-organ.
 4. The method of claim 1, wherein said genetically modified dermal micro-organ is 10-60 mm in length.
 5. The method of claim 4, wherein said genetically modified dermal micro-organ is 20-40 mm in length.
 6. The method of claim 1, wherein said dermal micro-organ includes at least part of the cross-section of the dermis.
 7. The method of claim 1, wherein at least one dimension of the cross section of said genetically modified dermal micro-organ is 0.5-3.5 mm.
 8. The method of claim 1, wherein said implanting comprises implanting said genetically modified dermal micro-organ subcutaneously.
 9. The method of claim 1, wherein said implanting comprises implanting said genetically modified dermal micro-organ deeper in the body than subcutaneous implanting.
 10. The method of claim 1, wherein said dermal micro-organ is obtained from said subject.
 11. A method of delivering a recombinant factor VIII protein, or equivalent thereof, to a subject comprising implanting in the subject a genetically modified dermal micro-organ expressing at least a recombinant factor VIII protein or an equivalent thereof, wherein said dermal micro-organ is an explant of living tissue consisting essentially of a plurality of dermal components and lacking a complete epidermal layer and maintaining the micro-architecture and three-dimensional structure of the dermal tissue from which they are obtained, having dimensions selected so as to enable passive diffusion of adequate nutrients and gases to cells of said dermal micro-organ and diffusion of cellular waste out of said cells so as to minimize cellular toxicity and concomitant death due to insufficient nutrition and accumulation of waste in said dermal micro-organ, wherein at least some of said cells of said dermal micro-organ express and secrete at least a portion of said recombinant factor VIII protein or equivalent thereof.
 12. The method of claim 11, wherein said genetically modified dermal micro-organ further comprises an in vivo demarcation.
 13. The method of claim 12, wherein said in vivo demarcation comprises an ink or stain on the peripheral surface of said micro-organ, or a green fluorescent protein (GFP) gene or a luciferase reporter gene expressed by said micro-organ.
 14. The method of claim 11, wherein said genetically modified dermal micro-organ is 10-60 mm in length.
 15. The method of claim 14, wherein said genetically modified dermal micro-organ is 20-40 mm in length.
 16. The method of claim 11, wherein said dermal micro-organ includes at least part of the cross-section of the dermis.
 17. The method of claim 11, wherein at least one dimension of the cross section of said genetically modified dermal micro-organ is 0.5-3.5 mm.
 18. The method of claim 11, wherein said implanting comprises implanting said genetically modified dermal micro-organ subcutaneously.
 19. The method of claim 11, wherein said implanting comprises implanting said genetically modified dermal micro-organ deeper in the body than subcutaneous implanting.
 20. The method of claim 11, wherein said dermal micro-organ is obtained from said subject. 